Signal peptidase polypeptides of Staphylococcus aureus

Details Signal peptidase polypeptides of Staphylococcus aureus Signal peptidase polypeptides of Staphylococcus aureus

1999-06-25

2001-10-09

Smith, Lynette R. F. (Department: 1645)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S070100, C435S183000, C435S252300, C435S254100, C435S320100, C435S325000, C435S455000, C435S471000, C514S04400A, C536S023100, C536S023200, C536S023700

Reexamination Certificate

active

06300097

ABSTRACT:

FIELD OF THE INVENTION
This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of these polynucleotides and polypeptides; processes for making these polynucleotides and these polypeptides, and their variants and derivatives and antagonists of the polypeptides; and uses of these polynucleotides, polypeptides, variants, derivatives and antagonists. In particular, in these and in other regards, the invention relates to polynucleotides and polypeptides of spsB, hereinafter referred to as “spsB”.
BACKGROUND OF THE INVENTION
The majority of proteins that are translocated across one or more membranes from the site of synthesis are initially synthesized with an N-terminal extension known as a signal, or leader, peptide (Wickner, W., et al, (1991).
Ann. Rev. Biochem.
60:101-124). Proteolytic cleavage of the signal sequence to yield the mature protein occurs during, or shortly after, the translocation event and is catalyzed in both prokaryotes and eukaryotes by enzymes known as signal, or leader, peptidases (SPases). The bacterial SPases are membrane proteins consisting of a single polypeptide anchored to the membrane by one (Gram-positive (G
+
) and Gram-negative (G
−
) bacteria) or two (G
−
bacteria) transmembrane sections. Predicted amino acid sequences of bacterial SPases show a high level of similarity and are known for
Escherichia coli
(Wolfe, P. B, et al, (1983)
J. Biol. Chem.
258:12073-12080),
Pseudomonas fluorescens
(Black, M. T., et al, (1992).
Biochem. J.
282:539-543),
Salmonella typhimurium
(van Dijl, J. M., et al, (1990).
Mol. Gen. Genet.
223:233-240),
Haemophilus influenzae
(Fleischmann, R. D., et al, (1995).
Science
269:496-512),
Phormidium laminosum
(Packer, J. C., et al, (1995).
Plant Mol. Biol.
27:199-204. K. Cregg, et al: Signal peptidase from
Staphylococcus aureus
Manuscript JB765-96),
Bradyrhizobium japonicum
(Müller, P., et al, (1995).
Mol. Microbiol.
18:831-840),
Rhodobacter capsulatus
(Klug, G., et al, (1996). GenBank entry, accession number 268305),
Bacillus subtilis
(two chromosomal and two of plasmid origin (Akagawa, et al, (1995)
Microbiol.
141:3241-3245; Meljer, W. J. J., et al, (1995).
Mol. Microbiol.
17:621-631; van Dijl, J. M., et al, (1992).
EMBO J.
II:2819-2828),
Bacillus licheniformis
(Hoang, V., et al, (1993). Sequence P42668 submitted to emb1/genbank/ddbj data banks.),
Bacillus caldotyricus
(van Dijl, J. M. (1993). Sequence p41027, submitted to embl1/genbank/ddbj data banks),
Bacillus amyloliquifaciens
(two chromosomal genes) (Hoang, V. and J. Hofemeister. (1995).
Biochim. Biophys. Acta
1269:64-68; van Dijl, J. M. (1993). Sequence p41026, submitted to emb1/genbank/ddbj data banks) and a partial sequence has been reported for
Bacillus pumilis
(Hoang, V. and J. Hofemeister. (1995).
Biochim. Biophys. Acta
1269:64-68). These enzymes have been collectively defined as type-1 signal pepidases (van Dijl, J. M., et al, (1992).
EMBO J.
II11:2819-2828). Although the amino acid sequences of fifteen bacterial SPases (and a sixteenth partial sequence) have now been reported, the best studied examples are leader peptidase (LPase or LepB) from
E. coli
and a SPase from
B. subtilis
(SipS).
It has been demonstrated that LPase activity is essential for cell growth in
E. coli.
Experiments whereby expression of the lepB gene, encoding LPase, was regulated either by a controllable ara promoter (Dalbey, R. E. and Wickner. 260:15925-15931) or by partial deletion of the natural promoter (Date, T. (1983).
J. Bacteriol.
154:76-83) indicated that minimization of LPase production was associated with cessation of cell growth and division. In addition, an
E. coli
strain possessing a mutated lepB gene (
E. coli
IT41) has been shown to have a drastically reduced growth rate and display a rapid and pronounced accumulation of preproteins when the temperature of the growth medium is elevated to 42° C. (Inada, T., et al, (1988).
J. Bactericol.
171:585-587). These results infer that there is no other gene product in
E. coli
that can substitute for LPase and that lepB is a single-copy gene in the
E. coli
chromosome. This is in contrast to at least two species within the G
+
Bacillus genus,
B. subtilis
and
B. amyloliquifaciens.
It is known that there are at least two homologous SPase genes in each of these Bacillus species. The sipS gene can be deleted from the chromosome of
B. subtilis
168 without affecting cell growth rate or viability under laboratory conditions to yield a mutant strain that can still process pre&agr;-amylase (K. M. Cregg and M. T. Black, unpublished). A putative SPase sequence (Akagawa, et al, (1995)
Microbiol.
141:3241-3245) may be the gene-product responsible for this activity and/or
B. subtilis
may harbor more than two SPase genes. Two or more genes encoding distinct SPase homologues reside on the chromosome of the closely related species
B. amyloliquifaciens
(Hoang, V. and J. Hofemeister. (1995).
Biochim. Biophys. Acta
1269:64-68) and there is evidence to suggest that
B. Japonicum
may possess more than one SPase (Müller, P., et al, (1995).
Mol. Microbiol.
18:831-840; Müiller, P., et al, (1995).
Planta
197:163-175). Although SPase sequences from seven genera of G+ bacteria are now known, only the single Bacillus genus amongst the G+ eubacteria has been investigated with respect to SPase characteristics. It was therefore considered of interest to determine whether a G+ eubacterium that, unlike
B. subtilis
and
B. amyloliquifaciens,
is not known for exceptional secretion activity has genes encoding more than one SPase with overlapping substrate specificities or whether it resembles
E. coli
and
H. influenzae
(and possibly other G− eubacteria)more closely in that it has a single SPase gene. The recent publication of the entire genomic sequence of the obligate G+-like intracellular bacterium
Mycoplasma genitalium
also reveals an interesting feature relating to heterogeneity amongst SPases (Fraser, C. M., et al, (1995).
Science
270:397-403). Inhibitors of
E. coli
LPase have bean reported (Allsop, A. E., et al, 1995.
Bioorg,
&
Med. Chem. Letts.
5:443-448).
Evidence has accumulated to suggest that LPase belongs to a new class of serine protease that does not utilize a histamine as a catalytic base (Black, M. T., et al, (1992).
Biochem. J.
282:539-543; Sung, M. and R. E. Dalbey. (1992).
J. Biol. Chem
267:13154-13159) but may instead employ a lysine side-chain to fulfill this role (Black, M. T. (1993).
J. Bacteriol.
175:4957-4961; Tschantz, W. R., et al, (1993)
J. Biol. Chem.
268:27349-27354). These observations and comparisons with Lex A from
E. coli
led to the proposal that a serine-lysine catalytic dyad, similar to that thought to operate during peptide bond hydrolysis catalyzed by LexA (Slilaty, S. N. and J. Little. (1987).
Proc. Natl. Acad. Sci. USA
84:3987-3991), may operate in LPase (Black, M. T. (1993).
J. Bacteriol.
175:4957-4961). Similar observations have since been made for SPase from
B. subtilis
(van Dijl, J. M., et al, (1995).
J. Biol. Chem.
270:3611-3618) and for the Tsp periplasmic protease from
E. coli
(Keiler, K. C. and R. T. Sauer. (1995).
Biol. Chem.
270:28864-28868); the similarities of SipS to LexA have been suggested to extend to several regions of primary structure (van Dijl, J. M., et al, (1995).
J. Biol. Chem.
270:3611-3618). The serine and lysine residues (90 and 145 respectively in
E. coli
LPase numbering) known to be critical for catalytic activity in both
E. coli
LPase (Black, M. T. (1993).
J. Bacteriol.
175:4957-4961; Tschantz, W. R., et al, (1993)
J. Biol. Chem.
268:27349-27354) and
B. subtilis
SPase (van Dijl, J. M., et al, (1995).
J. Biol. Chem.
270:3611-3618) and thought to form a catalytic dyad are both conserved in the
S. aureus
protein SpsB (S36 and K77). In addition, the aspartate at position 155 (280 in
E. coli
LPase numbering) is also conserved (this residue appears important for activity

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