Secretion and processing of staphylococcal nuclease by Bacillus subtilis

Miller, Jim; Kovacevic, Steven; Veal, L E

doi:10.1128/jb.169.8.3508-3514.1987

Cited by 33 publications

(31 citation statements)

References 29 publications

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“…glutamicum is one residue shorter than nuclease A of S. aureus Foggie (3), and the A form produced by B. subtilis is even an amino acid shorter (21). Heterogeneity of the amino terminus of S. aureus nuclease A has also been observed (19).…”

Section: Discussionmentioning

confidence: 95%

“…For C. glutamicum and S. aureus, the amino termini of the B forms of nuclease have been determined and were found to be identical (reference 5 and this report). For B. subtilis, the B form was not present in steady-state cultures, but an approximately 20-kDa protein comigrating with SNase B was found to be the first nuclease form released to the medium by this host organism too (21). Thus, primary processing of the SNase precursor seems to occur at the same site, which is a typical signal peptidase cleavage site (Ala-Asn-Ala l ), in all these organisms (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum

1992

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The gene for staphylococcal nudease (SNase), an extracellular enzyme of Staphylococcus aureus, was introduced into Corynebacterium gbiamicum. The heterologous gene was expressed in this host organism, and SNase was efficiently exported to the culture medium. Amino-terminal sequencing of SNase secreted by C. glutamicum revealed that the signal peptide was apparently cleaved off at precisely the same position as in the original host, S. aureus. As with S. aureus, a second smaller form of SNase (A form), whose appearance is presumably the result of a secondary processing step, was found in the culture medium of the recombinant C. glutamicum strain. The A form was one residue shorter than the mature nuclease A produced by S. aureus. Variation of the sodium chloride concentration in the growth medium had a marked influence on the location and the processing of SNase by C. glutamicum. In a complex growth medium containing 4% sodium chloride, SNase was exclusively located in the supernatant, but a significant amount of the enzyme remained cell associated if the strain was grown in a low-salt medium. Also, high salt concentrations seemed to inhibit processing of the high-molecular-weight form of SNase (B form) to the smaller A form. Similarities and differences in the export and modes of processing of SNase by three different, nonrelated gram-positive host organisms are discussed. Finally, a versatile Escherichia coli-C. gluamicum tae-lacl expression shuttle vector was constructed. With this vector, it was possible to achieve isopropyl-o-D-galactopyranoside (ITG)-inducible overexpression and secretion of SNase in C. gtamicum, whereby the expression level was dependent on the concentration of the inducer.Corynebacterium glutamicum is a gram-positive bacterium with a high G+C content of DNA and is closely related to other so-called "amino acid-producing corynebacteria" (12). These organisms are used for the industrial production of certain amino acids. Recent progress in the development of more sophisticated genetic tools for C glutamicum makes this species an interesting alternative gram-positive cloning host. Compared with other established high-G+C grampositive hosts such as certain Streptomyces species, C. glutamicum has some advantages; e.g., there are no complex cellular differentiation steps such as mycelium formation or sporulation during growth. Also, C. glutamicum does not have the disadvantage of seVere genetic instabilities, as are known to occur in Streptomyces species. One major advantage over low-G+C gram-positive hosts such as Bacillus species or Staphylococcus species is the rather broad acceptance of heterologous expression signals, including gram-negative promoters (23), observed for C. glutamicum ("Brevibacterium lactoferinentum").It is generally accepted that gram-positive bacteria are superior to other bacteria as protein secreters, the most prominent examples being certain Bacillus species. Nevertheless, knowledge about protein export by gram-positive bacteria is still very poor. This is especially ...

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum

1992

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“…The enzyme is large enough that it cannot rapidly diffuse through the cell wall. Small enzymes, such as staphylococcal nuclease (molecular weight, 16,800) are not impeded by the wall (25). Presumably, the LVS is passively carried through the wall to the cell surface as peptidoglycan-teichoic acid is laid down on the inner face of the wall (2, 8,19,21,26,28,29).…”

Section: Discussionmentioning

confidence: 99%

Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis

et al. 1993

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Bacterial metabolism excretes protons during normal metabolic processes. The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents. Calculations by Koch (J. Theor. Biol. 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH. That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments. When cultures of B. subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the ceUs bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO22+) ions and were readily agglutinated by cationized ferritin. In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (Cro42-). Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth. The composite interpretation of the foregoing observations suggests that the wail is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions. This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium.During chemiosmosis, protons are extruded through the cytoplasmic membrane and acidify a narrow region near the membrane. The protons subsequently return to the cytoplasm by mechanisms that extract work in the form of ATP creation or powering transport events. While outside the cell membrane, the local pH may be reduced by 3 to 4 U (17). It can be shown by physicochemical calculations based on the Debye-Huckel and Gouy-Chapman theories that the pH lowering is only a distance of a few nanometers if no cations are being pumped into the cell, but because potassium is needed in quantity by the growing cell the pH may be lowered throughout the 25-nm thickness of the gram-positive wall. The first evidence that this had important consequences for the cell was the study of Jolliffe et al. (12) which showed the activities of wall autolysins of Bacillus subtilis were increased (i.e., no longer inhibited) when chemiosmosis was blocked by adding proton conductors. The present paper brings two additional kinds of evidence on the influence of proton extrusion on wall physiology. The first comes from experiments showing that when the cell is metabolizing and extruding protons, positively charged probes (Cr3+, UO22+, cationized ferritin) display a weak affinity for the wall, whereas a negatively charged probe (CrO42-) binds the bacteria with a relatively high affinity. In contrast, when the proton motive force is dissipated, the positively charged probes readily complex with the cell surface, whereas the nega...

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“…The nuc gene encodes a preproprotein. The active 168-amino-acid proprotein, called NucB, is matured by several bacteria to form active NucA (17,22,23,25,38). Proprotein processing of NucB to NucA is mediated by the cell surface housekeeping proteinase HtrA in L. lactis (31).…”

Section: Discussionmentioning

confidence: 99%

Design of a Protein-Targeting System for Lactic Acid Bacteria

et al. 2001

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We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (ϳ95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.

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Secretion and processing of staphylococcal nuclease by Bacillus subtilis

Cited by 33 publications

References 29 publications

Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum

Expression, secretion, and processing of staphylococcal nuclease by Corynebacterium glutamicum

Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis

Design of a Protein-Targeting System for Lactic Acid Bacteria

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