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At least 19 recordsLinked to original sources
PubMed1996

Periplasm, periplasmic spaces, and their relation to bacterial wall structure: novel secretion of selected periplasmic proteins from Pseudomonas aeruginosa.

A brief overview of thin sections of cryopreserved walls from select eubacteria will be presented to suggest that all bacteria have functional periplasms, but that these are not necessarily confined to a periplasmic space such as found in typical gram-negative bacteria. Pseudomonas aeruginosa contains many components in its periplasmic space, some of which are required for infection. Throughout its growth cycle, P. aeruginosa blebs-off membrane vesicles that can possess DNA, endotoxin, phospholipase, protease, hemolysin, alkaline phosphatase, and autolysin, each of which must have a molecular phase that resides in the periplasm. These membrane packets make good delivery systems to convey these components to other bacteria and, possibly, tissue. Aminoglycoside antibiotics, such as gentamicin, produce a serious perturbation on the bacterium's surface (separate from the ribosomal effect), which contributes to the killing of the microorganism. Antibiotics such as this increase the size and number of the membrane blebs, which could contribute to septic shock of patients under drug therapy.

Aminoglycosides↗
PubMed1998

Selection for a periplasmic factor improving phage display and functional periplasmic expression.

The efficiency of both phage display in Escherichia coli and periplasmic expression of recombinant proteins may be limited by the same periplasmic folding steps. To search for E. coli factors that improve the efficiency of both procedures, a library of E. coli proteins was coexpressed in a phagemid vector that contained a poorly folding single-chain Fv antibody (scFv) fragment fused to g3p. We enriched, by panning for antigen binding, those phagemids in which the amount of displayed scFv is highest. We thus identified the periplasmic protein Skp/OmpH/HlpA as improving phage display of a wide range of scFv fragments. This occurs as a result of an increase in the amount of hybrid protein displayed on the phage. Coexpression of skp also increases the functional yield of scFv fragments when expressed by secretion to the periplasm.

Bacterial Proteins↗
PubMed1997

ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping.

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20-24 residue peptides of FhuB by determining the activity of beta-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA...G (in FhuB DTA ...G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.

Adenosine Triphosphate↗
PubMed1996

Periplasmic location of the pesticin immunity protein suggests inactivation of pesticin in the periplasm.

The pesticin activity and immunity genes on plasmid pPCP1 of Yersinia pestis were sequenced. They encoded proteins of 40 kDa (pesticin) and 16 kDa (immunity protein); the latter was found in the periplasm. The location of the immunity protein suggests that imported pesticin is inactivated in the periplasm before it hydrolyzes murein. Pesticin contains a TonB box close to the N-terminal end that is identical to the TonB box of colicin B. The DNA sequences flanking the pesticin determinant were highly homologous to those flanking the colicin 10 determinant. It is proposed that through these highly homologous DNA sequences, genes encoding bacteriocins may be exchanged between plasmids by recombination. In the case of pesticin, recombination may have destroyed the lysis gene, of which only a rudimentary fragment exists on pPCP1.

Amino Acid Sequence↗
PubMed2000

Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin.

Disulfide bond formation in Escherichia coli is a catalyzed reaction accomplished by DsbA. We found that null mutations in a new porin gene, ompL, allowed a total bypass of the DsbA requirement for protein oxidation. These mutations acted as extragenic null suppressors for dsbA, and restored normal folding of alkaline phosphatase and relieved sensitivity to dithiothreitol. ompL dsbA double mutants were completely like wild-type mutants in terms of motility and lack of mucoidy. This suppression was not dependent on DsbC and DsbG, since the oxidation status of these proteins was unaltered in ompL dsbA strains. Purified OmpL allowed diffusion of small solutes, including sugars, but the suppression was not dependent on the carbon sources used. Suppression by ompL null mutations required DsbB, leading us to propose a hypothesis that DsbB oxidizes yet unidentified, low-molecular-weight redox agents in the periplasm. These oxidized agents accumulate and substitute for DsbA if their leakage into the medium is prevented by the absence of OmpL, presumed to form a specific channel for their diffusion.

Amino Acid Sequence↗
PubMed1990

Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha. The periplasmic enzyme catalyzes the first step in aerobic denitrification.

The unusual ability of Thiosphaera pantotropha to catalyze respiratory nitrate reduction under aerobic conditions is shown to correlate with the activity of a periplasmic nitrate reductase that is expressed under both aerobic and anaerobic growth conditions. The organism also synthesizes, but only under anaerobic conditions, a membrane-bound nitrate reductase which resembles the corresponding enzyme in Paracoccus denitrificans in respect of both catalytic properties and inhibition of activity in intact cells in the presence of oxygen.

Aerobiosis↗
PubMed1993

Isolation of the periplasm of Neisseria gonorrhoeae.

The periplasm of Neisseria gonorrhoeae should be similar to other Gram-negative bacteria, but no published reports confirm this assumption. We used a periplasmic isolation procedure developed in Escherichia coli to release the periplasmic contents of N. gonorrhoeae. The resultant periplasmic extract lacked lipopolysaccharide, protein markers of inner or outer membranes, surface-radiolabelled protein components, or ribosomal proteins. The periplasmic extract contained a single haem protein believed to be a c-type cytochrome known to exist in the periplasm of other Gram-negative species, and retained significant alkaline phosphatase activity. The dominant protein species released in the periplasmic extract was the gonococcal homologue of elongation factor Tu, a major component released in similar periplasmic extracts of E. coli. These data showed that the extraction procedure selectively released periplasmic components and that the gonococcal periplasm was comparable to that of E. coli. Further analysis of the gonococcal periplasm may provide important insights into the physiology of this pathogen of humans.

Alkaline Phosphatase↗
PubMed1991

The bent-end morphology of Treponema phagedenis is associated with short, left-handed, periplasmic flagella.

Treponema phagedenis Kazan 5 is a spirochete with multiple periplasmic flagella attached near each end of the cell cylinder. Dark-field microscopy revealed that most of the cell is right-handed (helix diameter, 0.23 micron; helix pitch, 1.74 microns), and the ends appear bent. These ends could move and gyrate while the central part of the cell remained stationary. The present study examines the basis for the bent-end characteristic. Motility mutants deficient in periplasmic flagella were found to lack the bent ends, and spontaneous revertants to motility regained the periplasmic flagella and bent-end characteristic. The length of the bent ends (2.40 microns) was found to be similar to the length of the periplasmic flagella as determined by electron microscopy (2.50 microns). The helix diameter of the bent ends was 0.57 micron, and the helix pitch of the bent ends was 1.85 microns. The periplasmic flagella were short relative to the length of the cells (15 microns) and, in contrast to the reports of others, did not overlap in the center of the cell. Similar results were found with T. phagedenis Reiter. The results taken together indicate that there is a causal relationship between the bent-end morphology and the presence of short periplasmic flagella. We report the first three-dimensional description of spirochete periplasmic flagella. Dark-field microscopy of purified periplasmic flagella revealed that these organelles were left-handed (helix diameter, 0.36 microns; helix pitch, 1.26 microns) and only 1 to 2 wavelengths long. Because of a right-handed cell cylinder and left-handed periplasmic flagella along with bent ends having helix diameters greater than those of either the cell cylinder or periplasmic flagella, we conclude that there is a complex interaction of the periplasmic flagella and the cell cylinder to form the bent ends. The results are discussed with respect to a possible mechanism of T. phagedenis motility.

Cell Movement↗
PubMed2002

Direct measurement of free Ca(2+) shows different regulation of Ca(2+) between the periplasm and the cytosol of Escherichia coli.

As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal. However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect. Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place. Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli. Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence. Cytosolic free Ca(2+) was determined using aequorin alone. We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid. Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential. With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium. A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher. At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range. We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.

Aequorin↗
PubMed1997

Localization of alanyl aminopeptidase and leucyl aminopeptidase in cells of Pseudomonas aeruginosa by application of different methods for periplasm release.

Various methods for the isolation of periplasm were examined and compared with regard to the complete release of known periplasmic marker enzymes and the contamination of the periplasm by cytosol for Pseudomonas aeruginosa PAO1 as a significant Gram-negative test strain. The aim of the investigations was to clarify the exact localization of alanyl aminopeptidase (AAP) and leucyl aminopeptidase (LAP) of this microorganism and to evaluate these methods. The osmotic shock of NOSSAL and HEPPEL (1996) was the most effective method with the lowest contamination by the cytosolic marker enzyme malic enzyme, but some proteins, which are located near the inner side of the cytoplasmic membrane, can be released additionally into the periplasm. All other procedures like chloroform or polymyxin treatment, the magnesium chloride washing of intact bacteria and spheroblasting by lysozyme in the presence of EDTA or magnesium chloride resulted only in a partial, sometimes only very low release of periplasm. The periplasmic enzymes are bound either more by hydrophobic or more by ionic interactions to the cell envelope and show a different behaviour with the different releasing agents. These methods are useful for a further differentiation between really periplasmic protein, and those proteins, which were false positive found in periplasm as a result of the osmotic shock. Our results show that AAP from Pseudomonas aeruginosa is a periplasmic enzyme with hydrophobic interactions to the cytoplasmic membrane, corresponding to the early results of LAZDUNSKI and MURGIER for Escherichia coli (LAZDUNSKI et al. 1975a and b, MURGIER et al. 1977), and LAP is cytosolic, but located near the cytoplasmic membrane. The AAP is not a real amphipatic membrane protein, as could be demonstrated by phase separation experiments with Triton X-114.

Acid Phosphatase↗
PubMed2002

Identification of the periplasmic cobalamin-binding protein BtuF of Escherichia coli.

Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1998

Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake.

The inner membrane protein CcmC (CytA) of Pseudomonas fluorescens ATCC17400, which has homologues in several bacteria and plant mitochondria, is needed for the biogenesis of cytochrome c. A CcmC-deficient mutant is also compromised in the production and utilization of pyoverdine, the high-affinity fluorescent siderophore. A topological model for CcmC, based on the analysis of alkaline phosphatase fusions, predicts six membrane-spanning regions with three periplasmic loops. Site-directed mutagenesis was used in order to assess the importance of some periplasm-exposed residues, conserved in all CcmC homologues, for cytochrome c biogenesis, and pyoverdine production/utilization. Despite the conservation of the residues His-61, Val-62 and Pro-63 in the first periplasmic loop, and Leu-184, His-185 and Gln-186 in the third periplasmic loop, their simultaneous replacement with Ala only partially affected cytochrome c biogenesis and pyoverdine production/utilization. Simultaneous replacements of residues Trp-115 and Gly-116 in the second periplasmic loop substantially affected pyoverdine production/utilization but not cytochrome c production. An Ala substitution of Asp-127, in the second periplasmic loop, resulted in decreased production of cytochrome c, slower growth in conditions of anaerobiosis and reduced pyoverdine production. On the other hand, a mutation in Trp-126, also in the second periplasmic loop, totally suppressed the production of cytochrome c, whereas it had no effect on the production and utilization of pyoverdine. These results show a differential involvement of amino acid residues in periplasmic domains of CcmC in cytochrome c biogenesis and pyoverdine production/utilization.

ATP-Binding Cassette Transporters↗
PubMed2000

The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines.

The production of recombinant proteins in the periplasm of Escherichia coli can be limited by folding problems, leading to periplasmic aggregates. We used a selection system for periplasmic chaperones based on the coexpression of an E. coli library with a poorly expressing antibody single-chain Fv (scFv) fragment displayed on filamentous phage (Bothmann, H., and Plückthun, A. (1998) Nature Biotechnol. 16, 376-380). By selection for a functional antibody, the protein Skp had been enriched previously and shown to improve periplasmic expression of a wide range of scFv fragments. This selection strategy was now repeated with a library constructed from the genomic DNA of an skp-deficient strain, leading to enrichment of the periplasmic peptidylprolyl cis,trans-isomerase (PPIase) FkpA. Coexpression of FkpA increased the amount of fusion protein displayed on the phage and dramatically improved functional periplasmic expression even of scFv fragments not containing cis-prolines. In contrast, the coexpression of the periplasmic PPIases PpiA and SurA showed no increase in the functional scFv fragment level in the periplasm or displayed on phage. Together with the in vitro data in the accompanying paper (Ramm, K., and Plückthun, A. (2000) J. Biol. Chem. 275, 17106-17113), we conclude that the effect of FkpA is independent of its PPIase activity.

Bacteriophages↗
PubMed1996

Overall protein content and induced enzyme components of the periplasm of Bacillus subtilis.

Estimates for the overall protein content of the periplasm of Escherichia coli range from 4 to 16% of cellular protein. A cursory examination of known sources of contamination inherent to the methods employed for measurement leads to the conclusion that even the lower value may represent an overestimate of the periplasmic protein in E. coli. The protoplast supernatant fraction (PSF) of Bacillus subtilis defines operationally a potential periplasm, which, after correction for cytoplasmic contamination, yielded, in B. subtilis strains 168 and W23, calculated values of 9 and 3%, respectively, of cell protein as being periplasmic. 26 Among enzymes typically periplasmic in E. coli, at least two, RNases and a 5'-nucleotidase, were located in the B. subtilis periplasm. Compared to other cell fractions, RNase activity in the periplasm was associated with several protein bands forming a unique profile. Samples from all growth phases of cells cultured under phosphate-limitation and phosphate-excess revealed that a major part of both investigated activities was induced by phosphate depletion and located outside the plasma membrane. The current belief that a periplasm containing soluble enzymes does not exist in gram-positive bacteria is examined in light of the absence of an outer membrane permeability barrier, and of a clearly defined electron-transparent zone located between the plasma membrane and the cell wall of B. subtilis. Previous results of studies of protein secretion, and cell wall permeability, are reinterpreted by assuming that the thick charged cell wall of gram-positive bacteria can act as the outer permeability barrier, and as such be the functional equivalent of the outer membrane of gram-negative organisms.

Bacillus subtilis↗
PubMed1980

Involvement of periplasmic fibrils in motility of spirochetes.

Nonmotile (Mot-) strains of Spirochaeta aurantia and Spirochaeta halophila were isolated with a procedure involving mutagenesis of motile wild-type cells. Electron microscopy showed that a Mot- mutant strain of S. halophia possessed incomplete periplasmic fibrils, inasmuch as most or all of the filamentous portion of the periplasmic fibrils was absent. Some of the cells of this Mot-, fibril-defective mutant strain lacked the filamentous portion of the periplasmic fibrils and formed proximal hooks, whereas other cells appeared to have a very small segment of the filamentous portion of the periplasmic fibrils attached to the proximal hooks. Motile revertants were isolated repeatedly from cultures of the Mot-, fibril-defective mutant and from S. halophila Mot- mutants that completely lacked periplasmic fibrils. The motile revertants possessed periplasmic fibrils ultrastructurally indistinguishable from wild-type periplasmic fibrils. This study indicates that periplasmic fibrils play an essential role in the motility of spirochetes.

Cytoplasm↗