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PubMed2003

Foreign signal peptides can constitute a barrier to functional expression of periplasmic proteins in Haemophilus influenzae.

To study the periplasmic branch of iron (ferric ion) uptake systems in Gram-negative bacteria, genetic reconstitution experiments were initiated in Haemophilus influenzae involving exchange of the periplasmic iron-binding protein. The expression of many of the heterologous periplasmic ferric-binding proteins (FbpAs) was quite limited. Transformation experiments with the fbpA gene from Neisseria gonorrhoeae yielded two colony sizes with different phenotypic characteristics. The small colonies contained the intact N. gonorrhoeae fbpA gene and were deficient in utilization of transferrin iron. The large colonies contained hybrid H. influenzae/N. gonorrhoeae fbpA genes, were proficient in transferrin iron utilization and had enhanced levels of expression of FbpA. These hybrid genes included several that encoded the mature N. gonorrhoeae FbpA with the H. influenzae signal peptide. To more fully evaluate the effect of foreign signal peptides, a series of hybrid genes were prepared that exchanged the signal peptides from H. influenzae FbpA, N. gonorrhoeae FbpA and the TEM-1 beta-lactamase. The presence of the H. influenzae leader was required for functional expression of FbpAs and was shown to dramatically increase the level of beta-lactamase activity.

Amino Acid Sequence↗
PubMed1992

Disulphide bridge formation in the periplasm of Escherichia coli: beta-lactamase:: human IgG3 hinge fusions as a model system.

We report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human IgG3 hinge region (Hi) fused in-phase to the C-terminal end of bacterial TEM1 beta-lactamase (Bla). In the first fusion gene blahi, TEM1 beta-lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3' end of the beta-lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20 degrees C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin-resistance to the cells. These results indicate that beta-lactamase can efficiently direct the export of proteins fused to its C-terminus, and moreover, at least some of the exported fusion proteins must carry the beta-lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab')2-like dimer. The presence in the periplasm of 'oxidized' monomers (with intrachain disulphide bonds) as well as of several high-molecular-mass proteins, probably resulting from the association between monomers and other cysteine-rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.

Bacterial Proteins↗
PubMed1993

The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel.

The complete nucleotide sequence of the Escherichia coli nik locus, which has been suggested to encode the specific transport system for nickel, has been determined. It was found to contain five overlapping open reading frames that form a single transcription unit. Deduced amino acid sequence of the nik operon shows that its five gene products, NikA to NikE, are highly homologous to components of oligopeptide- and dipeptide-binding protein-dependent transport systems from several Gram-negative and Gram-positive species. NikA represents the periplasmic binding protein, NikB and NikC are similar to integral membrane components of periplasmic permeases, and NikD and NikE possess typical ATP-binding domains that suggest their energy coupling role to the transport process. Insertion mutations in nik genes totally abolished the nickel-containing hydrogenase activity under nickel limitation and markedly altered the rate of nickel transport. Taken together, these data support the notion that the nik operon encodes a typical periplasmic binding-protein-dependent transport system for nickel.

Adenosine Triphosphate↗
PubMed1986

Peptide uptake by Salmonella typhimurium. The periplasmic oligopeptide-binding protein.

The uptake of most peptides, including many peptide antibiotics, by the oligopeptide permeases of Escherichia coli and Salmonella typhimurium requires the function of a specific peptide-binding protein (the OppA protein) located within the periplasm. The OppA protein is the largest and most abundant periplasmic substrate-binding protein known and has an unusually broad substrate-binding specificity. The OppA protein has been purified to homogeneity and anti-OppA antibodies have been raised. The sequence of the OppA protein has been deduced from the nucleotide sequence of the oppA gene. This protein is unrelated to any other known periplasmic substrate-binding protein, either immunologically or in its amino acid sequence. The role of this protein in peptide transport is discussed.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha.

The periplasmic nitrate reductase of Thiosphaera pantotropha has been purified from a mutant strain (M-6) that overproduces the enzyme activity under anaerobic growth conditions. The enzyme is a complex of a 93-kDa polypeptide and a 16-kDa nitrate-oxidizable cytochrome c552. The complex contains molybdenum; a fluorescent compound with spectral features of a pterin derivative can be extracted. In contrast to the dissimilatory membrane-bound nitrate reductases, the periplasmic nitrate reductase shows high specificity for nitrate as a substrate and is insensitive to inhibition by azide. The 93-kDa subunit exhibits immunological cross-reactivity with the catalytic subunit of Rhodobacter capsulatus N22DNAR+ periplasmic nitrate reductase. Mass spectrometric comparisons of holo-cytochrome c552 and apo-cytochrome c552 demonstrated that the polypeptide bound two haem groups. Mediated redox potentiometry of the cytochrome indicated that the haem groups have reduction potentials (pH = 7.0) of approximately -15 mV and + 80 mV. The functional significance of these potentials is discussed in relation to the proposed physiological role of the enzyme as a redox valve.

Cell Membrane↗
PubMed1995

Purification and characterization of the periplasmic nickel-binding protein NikA of Escherichia coli K12.

The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel-binding protein NikA by cloning the nikA gene into an overexpression vector, pRE1. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion-exchange chromatography. N-terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel-binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence. NikA binds one atom of nickel/molecule of protein with a dissociation constant (Kd) of less than 0.1 microM. Other metals (cobalt, copper, iron) are bound at least 10-fold less tightly. The high specificity for Ni2+ is also demonstrated by high-performance immobilized-metal-ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 microM Ni2+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild-type strain MC4100 contains about 23,000 molecules of NikA/cell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar-dependent repellent chemotaxis.

Bacterial Proteins↗
PubMed1994

The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli.

The cydD gene of Escherichia coli encodes a protein which, together with the CydC protein, probably constitutes a heterodimeric, ABC-family membrane transporter, necessary for biosynthesis of the cytochrome bd quinol oxidase. Here, we demonstrate that a cydD mutant also fails to synthesise periplasmic c-type cytochrome(s), suggesting that the transporter exports haem or some other component involved in assembly of cytochromes that are found in, or exposed to, the periplasm. The CydDC system appears to be the first example of a transporter required for periplasmic cytochrome assembly processes requiring more than one type of haem. A mutant defective in trxB (adjacent to the cydDC operon, and encoding thioredoxin reductase) was unaffected in cytochrome c or bd assembly.

Adenosine Triphosphatases↗
PubMed1997

Characterization of a periplasmic peptidyl-prolyl cis-trans isomerase in Erwinia chrysanthemi.

The main determinant of the plant pathogen Erwinia chrysanthemi virulence is the production of extracellular enzymes, mainly pectate lyases. Adjacent to a pectate lyase encoding locus, we identified the gene rotA supposed to encode a folding catalyst. Overproduction of the protein and assay of activity using a synthetic substrate, confirmed that rotA encodes a periplasmic peptidyl-prolyl cis-trans isomerase. rotA disruption provokes no change in cell morphology, cell viability, growth rate or stability of the extracellular and periplasmic proteins. In addition, this mutation does not alter the activity of the pectate lyases, their stability in the periplasm during the transitory step of secretion or their recognition by the Out secretory system. rotA expression was followed using a rotA::uidA transcriptional fusion. Some environmental conditions, such as temperature variations and nitrogen starvation, modulate rotA expression. In contrast to the E. coli rotA gene, the E. chrysanthemi rotA possesses only one promoter and is not controlled by the CRP global regulator.

Base Sequence↗
PubMed1998

Procaryotic expression of single-chain variable-fragment (scFv) antibodies: secretion in L-form cells of Proteus mirabilis leads to active product and overcomes the limitations of periplasmic expression in Escherichia coli.

Recently it has been demonstrated that L-form cells of Proteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coli JM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

Animals↗
PubMed1992

Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein.

A Serpulina (Treponema) hyodysenteriae expression library was constructed in vector lambda ZAP and screened with a polyclonal antiserum raised against S. hyodysenteriae periplasmic flagella. A single immunoreactive plaque was chosen for further analysis. The recombinant phage from this plaque contained a gene encoding the 44-kDa protein that is on the outer layer (or sheath) of the periplasmic flagella. DNA sequence analysis showed that the gene encodes a protein of 320 amino acids. The protein is homologous to the flagellar sheath proteins of Treponema pallidum and Spirochaeta aurantia but not to any other flagellar proteins. We designated the cloned S. hyodysenteriae flagellar sheath protein gene flaA and the encoded protein FlaA. The 19 N-terminal amino acid residues of FlaA constitute a signal peptide that is cleaved from the protein before assembly onto the flagella in the periplasm. Amino acid residues 20 to 38 correspond to the N-terminal amino acid sequence of the native protein. Upstream from the gene, DNA motifs that are similar to the consensus Escherichia coli -35 and -10 promoter sequences and a ribosome binding site were identified. Downstream from the gene, two inverted repeat sequences that may serve as a rho-independent transcription termination signal are present.

Amino Acid Sequence↗
PubMed1996

Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery.

The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete. The complex PF are composed of multiple class A and class B polypeptides. Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro. To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S. hyodysenteriae which is deficient in both FlaA1 and FlaB1. To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed. The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively. The inactivated flagellar genes were introduced into S. hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis. Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant. These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins. The resulting flaA1 flaB1 mutant exhibited altered motility in vitro. Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy. The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S. hyodysenteriae in the gut. Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism. The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract. This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus.

Animals↗
PubMed1970

Biochemical and cytochemical evidence for the polar concentration of periplasmic enzymes in a "minicell" strain of Escherichia coli.

A number of "surface" enzymes of Escherichia coli (i.e., among those selectively released by osmotic shock) all displayed higher specific activities in extracts of minicells than in extracts of typical rod forms; these enzymes included alkaline phosphatase, cyclic phosphodiesterase, acid hexose monophosphatase, 5'-nucleotidase, and ribonuclease I. In addition, alkaline phosphatase, cyclic phosphodiesterase, and acid hexose monophosphatase were cytochemically localized to regions of minicell periplasm that resembled reactive polar enlargements of the periplasm in rod forms. In contrast, a number of "internal" cytoplasmic enzymes (inorganic pyrophosphatase, beta-galactosidase, glutamine synthetase, polynucleotide phosphorylase, and ribonuclease II) showed elevated or similar specific activities in extracts of rod forms versus extracts of minicells. A specific heat-labile inhibitor for 5'-nucleotidase, known to occur in the cytoplasm, also showed no enrichment in minicells. These findings indicate that the "surface" enzymes are segregated in vivo into the terminal minicell buds, possibly because these enzymes are concentrated in the polar enlargements of the periplasm in typical rod forms.

Alkaline Phosphatase↗
PubMed1981

Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12.

Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein.

Alkaline Phosphatase↗
PubMed1986

Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli.

The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-[3H]Gly. The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides. Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding. Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine. Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide. Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding. Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains.

Acetylation↗
PubMed1987

Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus.

The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities.

Amino Acid Sequence↗
PubMed1991

Molybdenum requirement for translocation of dimethyl sulfoxide reductase to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans.

Translocation of dimethyl sulfoxide (DMSO) reductase to the periplasmic space was studied in vivo with a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, using immunoblotting analysis and radioactive labeling. A polypeptide with an apparent molecular mass about 2,000 Da higher than that of DMSO reductase accumulated during induction of the reductase with DMSO. An uncoupler, carbonyl cyanide-m-chlorophenylhydrazone, inhibited the processing of the polypeptide after cells had been radioactively pulse-labeled with [35S]methionine. These results indicated that the higher-molecular-mass polypeptide was the precursor form of DMSO reductase. The precursor form accumulated in either the cytoplasm or the membrane, whereas the mature form accumulated in the periplasmic space. The membrane-bound precursor was sensitive to proteinase K treatment from both the cytoplasmic and periplasmic sides of the membrane, indicating that the polypeptide binds to the membrane, exposing it to both the outer and inner surfaces of the cytoplasmic membrane. Processing of the precursor was hampered by removal of molybdate from the medium and was restored by its readdition. It was also inhibited by the addition of tungstate in the medium.

Biological Transport↗
PubMed1993

Characterization of a periplasmic 3':5'-cyclic nucleotide phosphodiesterase gene, cpdP, from the marine symbiotic bacterium Vibrio fischeri.

Vibrio fischeri, a marine bacterium that forms a bioluminescent symbiosis with certain fish and squids, exhibits the unusual attribute of growth on 3':5'-cyclic AMP (cAMP), apparently through the activity of a 3':5'-cyclic nucleotide phosphodiesterase (3':5'-CNP) with exceptionally high activity. The V. fischeri 3':5'-CNP is located in the periplasm, a novel cellular location for this enzyme in bacteria. To gain insight into the physiological function of this enzyme, we cloned the gene (designated cpdP) encoding it from V. fischeri MJ-1. This is the first bacterial 3':5'-CNP gene to be cloned. Sequencing and analysis of the 1.26-kb cpdP locus revealed a single open reading frame specifying a protein of 330 amino acid residues, including a 22-amino-acid leader peptide. The putative cpdP promoter contained a reasonable -10 promoter region (TATTAT) but contained no obvious -35 region; instead, a 12-bp inverted repeat (TTAAATATTTAA) occurred just upstream of this location. A possible rho-independent transcriptional terminator with a calculated free energy of -21.2 kcal.mol-1 (ca. -88.7 kJ.mol-1) followed the CpdP protein coding sequence. The predicted subunit molecular weight of 33,636 for the mature CpdP protein (36,087 less 2,451 for the leader peptide) was consistent with the molecular weight of 34,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the CpdP protein exhibited 30.3% identity with that of the low-affinity 3':5'-CNP (PDE1) of Saccharomyces cerevisiae and 33.6% identity with that of the extracellular 3':5'-CNP of Dictyostelium discoideum. The residue identities clustered in two regions, residues 100 to 146 and 238 to 269, which contained 30 of the 33 amino acids conserved in all three proteins, 4 of which were histidines. A gene replacement mutant of V. fischeri MJ-1 containing a 0.45-kb BglII deletion within the cpdP gene lacked periplasmic 3':5'-CNP activity and did not grow on cAMP, confirming for V. fischeri the relationship among cpdP, synthesis of the periplasmic 3':5'-CNP, and growth on cAMP. The mutant exhibited no obvious sensitivity to high extracellular concentrations of cAMP (5 and 10 mM), suggesting that the enzyme does not play a role in defense against extracellular cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16.

Alcaligenes eutrophus H16 shows three distinct nitrate reductase activities (U. Warnecke-Eberz and B. Friedrich, Arch. Microbiol. 159:405-409, 1993). The periplasmic enzyme, designated NAP (nitrate reductase, periplasmic), has been isolated. The 80-fold-purified heterodimeric enzyme catalyzed nitrate reduction with reduced viologen dyes as electron donors. The nap genes were identified in a library of A. eutrophus H16 megaplasmid DNA by using oligonucleotide probes based on the amino-terminal polypeptide sequences of the two NAP subunits. The two structural genes, designated napA and napB, code for polypeptides of 93 and 18.9 kDa, respectively. Sequence comparisons indicate that the putative gene products are translated with signal peptides of 28 and 35 amino acids, respectively. This is compatible with the fact that NAP activity was found in the soluble fraction of cell extracts and suggests that the mature enzyme is located in the periplasm. The deduced sequence of the large subunit, NAPA, contained two conserved amino-terminal stretches of amino acids found in molybdenum-dependent proteins such as nitrate reductases and formate dehydrogenases, suggesting that NAPA contains the catalytic site. The predicted sequence of the small subunit, NAPB, revealed two potential heme c-binding sites, indicating its involvement in the transfer of electrons. An insertion in the napA gene led to a complete loss of NAP activity but did not abolish the ability of A. eutrophus to use nitrate as a nitrogen source or as an electron acceptor in anaerobic respiration. Nevertheless, the NAP-deficient mutant showed delayed growth after transition from aerobic to anaerobic respiration, suggesting a role for NAP in the adaptation to anaerobic metabolism.

Alcaligenes↗