Biomedical subjects

J M van Dijl

Publications and source records attributed to J M van Dijl.

At least 19 recordsLinked to original sources

PubMed1997

Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities. Constitutive and temporally controlled expression of different sip genes.

Most biological membranes contain one or two type I signal peptidases for the removal of signal peptides from secretory precursor proteins. In this respect, the Gram-positive bacterium Bacillus subtilis seems to be exceptional, because it contains at least four chromosomally-encoded type I signal peptidases, denoted SipS, SipT, SipU, and SipV. Here, we report the identification of the sipT and sipV genes, and the functional characterization of SipT, SipU, and SipV. The four signal peptidases have similar substrate specificities, as they can all process the same beta-lactamase precursor. Nevertheless, they seem to prefer different pre-proteins, as indicated by studies on the processing of the pre-alpha-amylase of Bacillus amyloliquefaciens in strains lacking SipS, SipT, SipU, or SipV. The sipU and sipV genes are constitutively transcribed at a low level, suggesting that they are required for processing of (pre-)proteins secreted during all growth phases. In contrast, the transcription of sipS and sipT is temporally controlled, in concert with the expression of the genes for most secretory proteins, which suggests that SipS and SipT serve to increase the secretory capacity of B. subtilis. Taken together, our findings suggest that SipS, SipT, SipU, and SipV serve different functions during the exponential and post-exponential growth phase of B. subtilis.

Amino Acid Sequence↗

PubMed1997

Molecular cloning and characterisation of the ribC gene from Bacillus subtilis: a point mutation in ribC results in riboflavin overproduction.

A mutation leading to roseoflavin resistance and deregulated riboflavin biosynthesis was mapped in the genome of the riboflavin-overproducing Bacillus subtilis strains RB52 and RB50 at map position 147 degrees. The chromosomal location indicates that the deregulating mutation in RB52 and RB50 is an allele of the previously identified ribC mutation. We cloned the ribC gene and found that it encodes a putative 36-kDa protein. Surprisingly, RibC has significant sequence similarity to flavin kinases and FAD synthases from various other bacterial species. By comparing the deduced amino acid sequence of RibC from the wild-type parent strain of RB50 with the RibC sequence from the riboflavin-overexpressing RB50 mutant we identified a point mutation that resulted in a Gly to Ser exchange in the C-terminal region of the product.

Amino Acid Sequence↗

PubMed1997

The chemistry and enzymology of the type I signal peptidases.

The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane-bound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.

Bacterial Proteins↗

PubMed1997

ATP-dependent proteases that also chaperone protein biogenesis.

The ATP-dependent proteases Clp and FtsH from bacteria, as well as mitochondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. The coordination of such processes with selective proteolysis may function in the quality control of protein biogenesis.

ATP-Dependent Proteases↗

PubMed1996

Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon.

Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein assembly, but not in degradation, were suppressed by overproduction of yeast mitochondrial Lon, an ATP-dependent serine protease. Suppression by Lon was enhanced by inactivation of the proteolytic site and was prevented by mutation of the ATP-binding site. It is suggested that the mitochondrial proteases Lon, Afg3p, and Rca1p can also serve a chaperone-like function in the assembly of mitochondrial protein complexes.

ATP-Dependent Proteases↗

PubMed1996

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids in Escherichia coli.

A system is described that enables the cloning of genes specifying detrimental proteins in Escherichia coli. The system is based on pUC plasmids and was developed for the expression of the Bacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression of csaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked the csaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of the csaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as the skc gene of Streptococcus equisimilis, which cannot be cloned in high copy numbers in E. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.

Bacterial Outer Membrane Proteins↗

PubMed1996

Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I.

Bacillus subtilis contains three chromosomally encoded type I signal peptidases (SipS, SipT and SipU), which remove signal peptides from secretory precursor proteins. In the present study the biological function of SipS and the regulation of its synthesis were analysed. Unlike the type I signal peptidase of Escherichia coli, SipS was essential neither for protein secretion nor viability of the cell. However, in the absence of SipS the rate of processing of several preproteins was reduced, and four of the seven major secreted proteins of B. subtilis were hardly detectable in the growth medium. Surprisingly, the processing of Bacillus amyloliquefaciens alpha-amylase and the secretion of at least two endogenous B. subtilis proteins was improved in the absence of SipS. These findings indicate that the substrate preference of SipS differs from that of SipT and SipU, and that SipS is an important factor determining the efficiency of protein secretion in B. subtilis. SipS is transcribed in a growth phase- and medium-dependent manner. In minimal medium, the growth phase-dependent transcription of sipS is controlled by the DegS-DegU two-component regulatory system, indicating that the expression of sipS is regulated by the same factors that control the expression of most genes for secreted degradative enzymes. Our observations suggest that B. subtilis can modulate its capacity and specificity for protein secretion through the controlled expression of sipS.

Bacillus subtilis↗

PubMed1996

The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis.

The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the Escherichia coli beta-galactosidase (lacZ) gene. Two processes affected the accumulation of pGP1 variants with deletions in the penP-lacZ region. First, divergent transcription from genes upstream of penP-lacZ increased pGP1 deletion frequencies up to about 10-fold. Second, the removal of the PenP signal peptide resulted in completely stable plasmids, indicating that the entry of the PenP fragment into the protein export pathway is an important factor in the instability of pGP1. On the basis of these results, we propose a model in which the temporary anchoring of the plasmid to the membrane through the cotranscriptional and cotranslational entry of PenP into the protein export pathway creates domains of local hypersupercoiling, which we assume to be targets for deletion formation.

Amino Acid Sequence↗

PubMed1995

Identification of the potential active site of the signal peptidase SipS of Bacillus subtilis. Structural and functional similarities with LexA-like proteases.

Signal peptidases remove signal peptides from secretory proteins. By comparing the type I signal peptidase, SipS, of Bacillus subtilis with signal peptidases from prokaryotes, mitochondria, and the endoplasmic reticular membrane, patterns of conserved amino acids were discovered. The conserved residues of SipS were altered by site-directed mutagenesis. Replacement of methionine 44 by alanine yielded an enzyme with increased activity. Two residues (aspartic acid 146 and arginine 84) appeared to be conformational determinants; three other residues (serine 43, lysine 83, and aspartic acid 153) were critical for activity. Comparison of SipS with other proteases requiring serine, lysine, or aspartic acid residues in catalysis revealed sequence similarity between the region of SipS around serine 43 and lysine 83 and the active-site region of LexA-like proteases. Furthermore, self-cleavage sites of LexA-like proteases closely resembled signal peptidase cleavage sites. Together with the finding that serine and lysine residues are critical for activity of the signal peptidase of Escherichia coli (Tschantz, W.R., Sung, M., Delgado-Partin, V.M., and Dalbey, R.E. (1993) J. Biol. Chem. 268, 27349-27354), our data indicate that type I signal peptidases and LexA-like proteases are structurally and functionally related serine proteases. A model envisaging a catalytic serine-lysine dyad in prokaryotic type I signal peptidases is proposed to accommodate our observations.

Amino Acid Sequence↗

PubMed1995

The endogenous Bacillus subtilis (natto) plasmids pTA1015 and pTA1040 contain signal peptidase-encoding genes: identification of a new structural module on cryptic plasmids.

Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis plasmids pTA1015 and pTA1040. It is composed of two genes: one specifies an unidentified protein with a putative signal peptide; and the other (sipP) specifies a functional type 1 signal peptidase (SPase). The homologous, but non-identical, sipP genes of the two plasmids are the first identified plasmid-specific SPase-encoding genes. With respect to structure and activity, the corresponding enzymes (denoted SipP) are highly similar to the chromosomally encoded SPase, SipP, of B. subtillis and several newly identified SPases of other bacilli. Our findings suggest that plasmid-encoded SPases have evolved because, of under certain conditions, SPase can be a limiting factor for protein secretion in B. subtilis.

Amino Acid Sequence↗

PubMed1993

In vitro assay for the Bacillus subtilis signal peptidase SipS: systems for efficient in vitro transcription-translation and processing of precursors of secreted proteins.

The signal peptidase (SPase) SipS of Bacillus subtilis is responsible for the processing of precursors of secreted proteins. It differs from the SPases of Gram-negative bacteria in structure and specificity. To assay the activity of SipS in vitro, two efficient transcription-translation systems for the synthesis of radio-labelled precursors were developed. The systems were completely derived from B. subtilis. Post-translational in vitro processing of pre-staphylokinase by SipS was demonstrated. SipS activity was stimulated in vitro by several non-ionic detergents, whereas it was not affected by a large variety of proteinase inhibitors. SipS shares the latter property with other SPases.

Bacillus subtilis↗

PubMed1992

Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases.

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta-lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.

Amino Acid Sequence↗

PubMed1992

Suppression of the growth and export defects of an Escherichia coli secA(Ts) mutant by a gene cloned from Bacillus subtilis.

A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42 degrees C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli secY24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.

Adenosine Triphosphatases↗

PubMed1992

Protein export elements from Lactococcus lactis.

Broad-host-range plasmids carrying alpha-amylase or beta-lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the export of the reporter proteins in Escherichia coli. Several of the selected export elements were also active in Bacillus subtilis and L. lactis, although the efficiencies depended strongly on the host organism and reporter gene used. The export elements AL9 and BL1 were highly efficient in L. lactis in the expression and secretion of at least two heterologous proteins (Bacillus licheniformis alpha-amylase and E. coli TEM-beta-lactamase). AL9 even permitted growth of this organism on starch as the sole carbon source. Nucleotide sequence analysis of five selected fragments indicated that these encode oligopeptides with the major characteristics of typical signal peptides. The putative expression signals had a limited similarity to previously described expression signals for E. coli, B. subtilis and L. lactis. Differences in both expression and export efficiency are likely to underlie the host-specific effects.

Amino Acid Sequence↗

PubMed1991

Lack of specific hybridization between the lep genes of Salmonella typhimurium and Bacillus licheniformis.

This paper describes an attempt to clone the Bacillus licheniformis lep gene, encoding signal peptidase, using the Salmonella typhimurium lep gene as a hybridization probe. Although a hybridizing fragment was obtained, DNA sequence analysis indicated that it did not contain the lep gene. Instead, the protein encoded by the cloned fragment showed similarity with a variety of L-asparaginases.

Amino Acid Sequence↗

PubMed1991

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli.

The effects of 25-fold overproduction of Escherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from the Bacillus subtilis chromosome and the mature part of TEM-beta-lactamase, were studied in E. coli. One precursor (pre[A2d]-beta-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[A13i]-beta-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature beta-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-beta-lactamase and pre(A2)-beta-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export in E. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.

Bacillus subtilis↗

PubMed1991

Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis.

The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half that produced in wild-type E. coli cells. The production of E. coli SPase I in B. subtilis was increased approximately fivefold by cloning the lep gene into a high-copy-number plasmid. The expression of E. coli SPase I in B. subtilis did not appear to increase the rate of processing of two hybrid secretory precursor proteins. Two observations may explain the failure of E. coli SPase I to stimulate processing of exported proteins in B. subtilis. First, the E. coli SPase I was apparently not exposed on the outside of the B. subtilis cytoplasmic membrane, indicating its incorrect insertion into the membrane. Second, in vitro processing studies, using cell-free extracts of B. subtilis producing E. coli SPase I, suggested that the enzyme was not active. A further outcome of this study was that conditions favouring processing of precursors by SPase I in cell-free extracts of E. coli did not favour processing by the corresponding enzyme in B. subtilis cell-free extracts. This suggests that significant differences exist between the two enzymes. The observation that antibodies directed against E. coli SPase I did not cross-react with B. subtilis membrane proteins supports this idea.

Amino Acid Sequence↗

PubMed1990

Molecular cloning of the Salmonella typhimurium lep gene in Escherichia coli.

A system is described which enabled the selection of a heterologous lep gene, encoding signal peptidase I, in Escherichia coli. It is based on complementation of an E. coli mutant, in which the synthesis of signal peptidase I can be regulated. With this system the lep gene of Salmonella typhimurium was cloned and the nucleotide sequence was determined. The S. typhimurium lep gene encodes a protein of 324 amino acids. Expression of the gene in the E. coli mutant resulted in suppression of growth inhibition and in the restoration of processing activity under conditions where synthesis of E. coli signal peptidase I was repressed. The cloned S. typhimurium signal peptidase I had an apparent molecular weight of 36,000 daltons, which is in agreement with the calculated molecular weight of 35,782 daltons. The system described for selection of the S. typhimurium lep gene may permit the cloning and expression of other heterologous signal peptidase I genes.

Amino Acid Sequence↗