Subcellular distribution of alkalophilic Bacillus penicillinase produced by Escherichia coli HB101 carrying pEAP31

Summary The extracellular production of alkalophilic Bacillus penicillinase by Escherichia coli HB101 carrying pEAP31 was dependent on the cultivation temperature. Extracellular production occurred only above 26°C. The penicillinase produced by the organism grown at lower temperatures accumulated in the periplasm of the cells. At high temperature, the penicillinase accumulated transiently in the periplasm and then was released gradually from the cells. The penicillinase that accumulated in the periplasm of the organism grown at low temperature could also be released by shifting to a high temperature.     

Probable detection of kil peptide derived from colicin E1 plasmid in the envelope fraction of Escherichia coli HB101 carrying pEAP31.

Escherichia coli carrying plasmid pEAP31 produces extracellularly alkalophilic Bacillus penicillinase encoded on the plasmid. The extracellular production has been suggested to be caused by activation of dormant colicin E1 kil gene. Two peptides that could be respectively precursor and mature products of colicin E1 kil gene were detected on an SDS/polyacrylamide gel. One of the peptides (Mr 4800), which was probably a precursor peptide, was detected in the inner-membrane fraction from the organism when envelope proteins were subjected to differential solubilization. The other (Mr 3500), which was a mature peptide, was detected in the outer-membrane fraction of the organism. The mature peptide was only detected in the envelope of cells releasing the penicillinase transiently accumulated in the periplasm into the culture medium.     

Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane.

Two plasmids containing the penicillinase gene of alkalophilic Bacillus sp. strain 170, pEAP1 and pEAP2, were constructed. Most of the penicillinase produced by Escherichia coli, which carried these plasmids, was found in the culture medium. This excretion is caused by the cloned DNA fragment which contains some component that changes the outer membrane of E. coli.     

Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9.

Most of the cloned penicillinase from alkalophilic Bacillus sp. strain 170 and alkaline phosphatase were released into the culture medium by Escherichia coli strains bearing plasmid pEAP1 or pEAP2 (T. Kudo, C. Kato, and K. Horikoshi, J. Bacteriol. 156:949-951, 1983). We analyzed the basis for excretion of periplasmic enzymes in the cells bearing these plasmids. Several experiments such as subcloning, insertion of a chloramphenicol acetyltransferase cartridge, and DNA sequencing were done. A dormant kil gene in plasmid pMB9 was expressed by a promoter of the inserted DNA fragment of alkalophilic Bacillus sp. strain 170, and as a result, the outer membrane of E. coli became permeable, allowing the proteins to be excreted without cell lysis.     

Construction of new excretion vectors: two and three tandemly located promoters are active for extracellular protein production from Escherichia coli

Summary In order to develop a more useful system for extracellular protein production from Escherichia coli, we have constructed the new excretion vectors pEAP82-1, pEAP82-2, and pEAP82-3. These vectors have, respectively, a single, double, and triple penicillinase promoter upstream of a penicillinase structural gene; E. coli HB101 carrying pEAP82-2 or pEAP82-3 produced respectively, about twice or three times as much penicillinase protein than that produced by E. coli carrying pEAP82-1, and 70% to 80% of the protein was excreted into the culture medium. The E. coli carrying pEAP82-3 was cultivated at various temperatures and it was observed that the optimum for extracellular penicillinase production was 30°–37°C. Using this multi-promoter excretion system, the amount of extracellular production of human growth hormone was increased by several fold as observed with penicillinase excretion.     

Construction of an excretion vector and extracellular production of human growth hormone from Escherichia coli

A new excretion vector, pEAP8, was constructed to develop an excretion system for Escherichia coli. This plasmid, derived from pEAP37, carried the weakly activated kil gene of plasmid pMB9 [Kobayashi et al., J. Bacteriol. 166 (1986) 728-732] and the penicillinase promoter and signal region of an alkalophilic Bacillus sp. to excrete foreign gene products. A gene for human growth hormone (hGH) was joined to this signal sequence through the HindIII site. The recombinant plasmid p8hGH1 thus constructed, was introduced into E. coli. The hybrid protein which was produced in E. coli carrying p8hGH1 was processed during transport through the inner membrane, with the mature hGH being excreted into the medium through the outer membrane which was made permeable by the action of the kil gene. The N-terminal amino acid sequence and the biological activity of the extracellular hGH were consistent with those of the authentic hGH.     

Construction of a secretion vector production of peptide hormones in Escherichia coli (extracellular production of calcitonin as fused protein)

A new secretion vector, pEAP84 which contained a unique restriction site (BglII) at the 3' end of the penicillinase gene to produce a fused protein, and the Ex-kil region to make the outer membrane permeable, was constructed from pEAP82. A recombinant plasmid p84h06, which contained a synthetic gene for human calcitonin with a cyanogen bromide cleavage site at the junction site of the fused protein, was constructed and introduced into Escherichia coli. The hybrid protein produced in E. coli carrying p84h06 was secreted into the culture medium. The amino acid composition of this product was consistent with that deduced from the DNA sequence. Mature calcitonin was obtained following cyanogen bromide cleavage of the fused protein.     

Cultivation conditions for extracellular production of penicillinase by Escherichia coli carrying pEAP31 on a semi-large scale

Summary Cultivation conditions for extracellular production of penicillinase on a semi-large scale were established by using Escherichia coli K-12 HB 101 carrying the plasmid pEAP31 with the penicillinase gene from alkalophilic Bacillus sp. no. 170. Extracellular production of the enzyme was affected by several parameters such as concentration of carbohydrates and Nacl, pH value of culture broth, culture temperature, culture volume and shaking speed of the cultivation flask. The organism produced a large amount of the enzyme in culture broth under the optimal conditions established. For example, 180 units/ml of the extracellular enzyme was produced when the organism was inoculated in 300 ml broth in a 500-ml volume cultivation flask and shaken at 30°C on a reciprocal shaker at 172 oscillations/min with 3.2-cm strokes.     

High-production mutant Claviceps purpurea 59 accumulating secoclavines

Abstract A new cloning vehicle, pEAP37 was constructed to develop the excretion system of Escherichia coli . This plasmid, derived from pEAP1, carried the penicillinase gene from an alkalophilic Bacillus sp. and the chloramphenicol acetyltransferase gene from pBR329 as selective markers. The Bacillus N-4 cellulase gene, Bacillus 1139 cellulase gene and Aeromonas sp. 212 xylanase L gene was inserted into pEAP37, and the distribution of the plasmid-encoded enzymes was analyzed. Most of these enzyme activities were found in the periplasmic space of E. coli when these extracellular enzyme genes were inserted into pBR322. On the other hand, most of these activities were observed in the culture medium when inserted into pEAP37.     

The penicillinase of Bacillus licheniformis is an outer membrane protein in Escherichia coli.

The cloned gene coding for Bacillus licheniformis penicillinase (penP) was introduced into Escherichia coli in a heat-inducible lambda Qam vector. After induction, significant amounts of penicillinase were synthesized in the new host. The cellular location of the penicillinase was found to be almost exclusively the outer membrane fraction of E. coli, and virtually no soluble penicillinase was found. According to sodium dodecyl sulfate-gel electrophoresis, the size of the penicillinase from E. coli was identical to that of the membrane-bound form of the B. licheniformis penicillinase. Gel filtration in the presence of Triton X-100 suggested that the penicillinase from E. coli had amphiphilic properties, as does B. licheniformis membrane penicillinase. These results show that the export of the penicillinase to the outer membrane of E. coli involves the cleavage of the signal peptide from the prepenicillinase, giving an outer membrane component indistinguishable from the membrane penicillinase of B. licheniformis.     

Accumulation of glyceride-modified pre-penicillinase of Bacillus licheniformis in Escherichia coli treated with globomycin

The membrane penicillinase of Bacillus licheniformis is a glyceride-cysteine lipoprotein whose NH2 terminus is analogous to the major outer membrane lipoprotein of Escherichia coli. When E. coli cells producing B. licheniformis penicillinase were treated with the antibiotic, globomycin, a precursor of the penicillinase, pre-penicillinase, accumulated in the cell. It could be immunoprecipitated with anti-penicillinase antibodies; it contained palmitate; and one of its two cysteine residues was modified by glycerol. The action of globomycin, probably indirectly, also activates protease which acts differently on the pre-penicillinase than does the signal peptidase. The results strongly indicate that the pre-penicillinase is processed by the globomycin-sensitive signal peptidase in E. coli, and the modification of precursor by lipid precedes removal of the signal peptide as it does with the membrane lipoproteins of E. coli.     

Penicillinase-releasing protease of Bacillus licheniformis: purification and general properties.

The membrane penicillinase of Bacillus licheniformis 749/C is a phospholipoprotein which differs from the exoenzyme in that it has an additional sequence of 24 amino acid residues and a phosphatidylserine at the NH2 terminus. In exponential-phase cultures, the conversion of membrane penicillinase to exoenzyme occurs at neutral and alkaline pH. An enzyme that will cleave the membrane penicillinase to yield the exoenzyme is present (in small amounts) in exponential-phase cells and is released during their conversion to protoplasts. The enzyme is found in the filtrate of a stationary-phase culture of the uninduced penicillinase-inducible strain 749 and has been purified to apparent homogeneity from this source. The protease has an approximate molecular weight of 21,500 and requires Ca2+ ions for stabilization. It has a pH optimum of 7.0 to 9.5 for hydrolysis of casein and for the cleavage of membrane penicillinase. Both activities are inhibited by diisopropylfluorophosphate; hence, the enzyme is a serine protease. This enzyme may be entirely responsible for the formation of exopenicillinase by this organism, since the other neutral and alkaline proteases of strain 749 have little, if any, activity in releasing exopenicillinase. The enzyme has been termed penicillinase-releasing protease.     

Characteristics of Penicillinase Secretion by Growing Cells and Protoplasts of Bacillus licheniformis

Cultures of the inducible penicillinase-producing strain 749 of Bacillus licheniformis, induced with small amounts of benzylpenicillin, synthesized penicillinase at a high rate for a short period, after which the rate of synthesis slowly declined. During the period of active synthesis, the rate of secretion, as a fraction of the level of cell-bound penicillinase (which is originally high), gradually decreased to a constant level. Chloramphenicol, at a concentration (40 mug/ml) which completely inhibited synthesis of penicillinase, partially inhibited secretion if added during the period of active synthesis. During the phase of reduced synthesis, chloramphenicol was without effect on secretion. Penicillinase secretion, by actively growing cultures of the constitutive penicillinase-producing mutant 749/C, was inhibited by 75% immediately after addition of chloramphenicol. The secretion of part of the penicillinase released during active growth is probably dependent on synthesis of penicillinase, but part of the secreted penicillinase can be released in the absence of synthesis. Protoplasts were obtained from which periplasmic penicillinase has been removed, and these protoplasts were capable of substantial growth and penicillinase synthesis without lysis. At pH 7.5, there was no net incorporation of penicillinase into the cell membrane; the enzyme released was almost entirely of the exo form and was roughly equivalent to the amount of new enzyme formed. At pH 6.0, there was some incorporation of penicillinase into the plasma membrane, and approximately half of the extracellular penicillinase was in the exo form; the remainder perhaps represented membrane fragments. In the presence of chloramphenicol, a small amount of penicillinase was released at pH 7.5 as the exo form; at pH 6.0, practically none was released. We suggest that, with the removal from protoplasts of the periplasmic penicillinase-containing particles, a restriction on secretion has been lifted.     

Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.     

Wild-Type Strain of Staphylococcus aureus Containing Two Genetic Linkage Groups for Penicillinase Production

Staphylococcus aureus strain 55C1, isolated from a patient in 1955, contained two genetic linkage groups for penicillinase formation. One was linked to genes that control resistance to cadmium and mercuric ions; it had properties of a plasmidborne gene. The other was not linked to resistance to these metal ions; it had properties of a chromosomal gene. Penicillinase formation by cells that contained either linkage group was inducible by penicillins. Induced penicillinase in cells that contained both linkage groups equalled the sum of that produced in cells containing each group singly. Exopenicillinase produced by cells containing either gene was serological type A. Constitutive penicillinase formation resulting from regulator gene mutations in either linkage group was repressed to differing extents by a wild-type determinant in the trans position. The genetic structure and the regulation of penicillinase formation in strain 55C1 resembled in general those for penicillinase linkage groups which Asheshov and Dyke described for diploid mutant strains of S. aureus PS 80. There were differences in detail, however.     

Vesicle penicillinase of Bacillus licheniformis: existence of periplasmic-releasing factor(s).

In earlier studies of the membrane-bound penicillinase of Bacillus licheniformis 749/C, the enzyme present in the vesicles that were released during protoplast formation and the enzyme retained in the plasma membrane of protoplasts appeared to differ (i) in their behavior on gel permeation chromatography in the presence or absence of deoxycholate and (ii) in their tendency to convert to the hydrophilic exoenzyme (Sargent and Lampen, 1970). We have now shown that these vesicle preparations contain a soluble, heat-sensitive enzyme(s) that is released along with the vesicles during protoplast formation. The enzyme will convert the vesicle penicillinase to a form that resembles exopenicillinase, and this conversion can be inhibited by deoxycholate under certain circumstances. Sedimentation of such vesicle preparations at 100,000 X g produces vesicles which contain penicillinase that behaves as the plasma membrane enzyme obtained from protoplasts. Exopenicillinases released by growing cells at pH 6.5 and by washed cells or protoplasts at pH 9.0 have the same NH2-terminal residues (lysine and some glutamic acid); in addition, the various release systems show a parallel sensitivity to inhibition by deoxycholate, quinacrine, chloroquine, and o-phenanthroline. The formation of exopenicillinase (by cleavage of the membrane-bound enzyme) may well be dependent on the action of the releasing enzyme.     

Relationship of a Wall-Associated Enzyme with Specific Layers of the Cell Wall of a Gram-Negative Bacterium

In untreated cells of the marine pseudomonad studied here, alkaline phosphatase was found to be located in the periplasmic space, at the cell surface, and in the medium into which it had been shed during growth. Washing in 0.5 M NaCl, which removed the loosely bound outer layer, caused a shift of periplasmic enzyme to the outer aspect of the double-track layer and released some of the cell surface-associated enzyme. When the double-track layer of the cell wall was partially deranged, large amounts of this cell wall-associated enzyme were released, and, when the double-track was removed from the cells to produce mureinoplasts, alkaline phosphatase was released into the menstruum. There was no significant association of the enzyme with the peptidoglycan layer of the cell wall, which is the outermost structure of the mureinoplast, and no association of the enzyme with the cytoplasmic membrane of these modified cells. This study has shown that alkaline phosphatase is specifically associated with the outer layers of the cell walls of cells of this organism and is retained within the cell wall by virtue of this association.     

Localization of Cell-bound Penicillinase in Bacillus licheniformis

When protoplasts are prepared from Bacillus licheniformis (strain 749/C, constitutive for penicillinase), approximately 60% of the cell-bound penicillinase is released. The remainder is retained by the protoplast and cannot be removed by washing. This release is specific, in that less than 7% of the cellular reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and alpha-glucosidase is liberated by the treatment. The freed penicillinase is excluded from G-200 Sephadex, and it is partially sedimented with a force of 65,000 x g for 20 hr. It is probably attached to characteristic tubular and vesicular structures with single-layered membranes that are comparable to structures previously described in intact penicillinase-forming cells. The specific activity of the organelle is more than six times that of twice washed peripheral membrane; furthermore, about 8% of the protein of the structure is penicillinase. At substrate concentrations (benzylpenicillin) of about one-fifth the K(m) value, whole cells show a slight permeability restriction, although this does not occur in isolated particles and protoplasts.     

Study on biological effect of La3+ on Escherichia coli by atomic force microscopy

The biological effects of rare-earth metal ions on the organism have been studied using La3+ as a probe ion and Escherichia coli cell as a target organism. Atomic force microscopy (AFM) studies reveal that La3+ substantially changes the structure of the outer cell membrane responsible for the cell permeability. Significant damages of the outer cell membrane are observed using scanning electron microscopy (SEM) after the introduction of La3+. In result, the cell becomes easily attacked by lysozyme. Moreover, inductively coupled plasma-mass spectrometry (ICP-MS) measurements show considerable amount of Ca2+ and Mg2+ in the supernatant from the La3+ exposed cells. It is proposed that La3+ can replace Ca2+ from the binding sites because of their close ionic radii and similar ligand specificities. Lipopolysaccharide (LPS), which forms the outer membrane of Gram-negative bacteria, could not serve as the cellular envelope steadily after Ca2+ and Mg2+ released from their binding sites on the LPS patches.     

Relation of Integrity of Outer Membrane with Its Protection from Oxygen in Anabaena Cell

Exposure of Anabaena 7120 cells to membrane perturbant such as EDTA or Tris (pH 8.0, 37℃, for 5–10 min) resulted in the release of outer membrane lipopolysaccharide and proteins from cells. After Tris treatment, the sensitivity of cells to crystal violet and detergents such as SDS and Triton x-100 increased and whole-cell alkaline phosphatase activity enhanced obviously, suggesting that the structure of outer membrane was modified and its permeability increased. At the same time. Tris was found to reduce nitrogen fixation activity of cells considerably in air, but not in anaerobic condition. Reconstitution of Tris-treated cells with released material might recover nitrogen-fixing activity of cells clearly, indicating that the structure of outer membrane is closely related to the protection of nitrogen fixation from oxygen. Although EDTA-treated cells released more lipopolysacharide and proteins than those of Tris-treated cells, the permeability of outer membrane and nitrogen-fixing activity were not influenced significantly. SDS-gel electrophoresis showed that Tris-treated cells released 3–4 specific polypeptides which were not present in the released material from EDTA-treated or water-treated cells. These experiments suggest that membrane perturbants-induced loss of outer membrane function is mediated through the modification of specific position in outer membrane.