*Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, the Netherlands;
Department of Biochemistry, University of Groningen, Groningen, the Netherlands;
and
Research Laboratories Alko Ltd., Helsinki, Finland
The recent sequencing of the genome of Rickettsia prowazekii, a eubacterium that is more closely related to mitochondria than to any other known prokaryote, has provided exciting new insights into the evolution of mitochondria and their genomes (Andersson et al. 1998
). Consistent with the endosymbiont hypothesis (Gray et al. 1989
; Gray 1993
; Yang et al. 1985
), many mitochondrial proteins appear to be functionally conserved in R. prowazekii and other eubacteria. An important exception concerns the proteins which make up the machinery for the import of mitochondrial proteins from the cytosol. In this study, we addressed the function of a eubacterial homolog of mitochondrial processing peptidases (MPPs), which remove targeting signals from proteins imported into the mitochondrial matrix compartment.
Most mitochondrial proteins are encoded by nuclear DNA and synthesized in the cytosol. Hence, they have to be imported into mitochondria, for which purpose they are synthesized with an amino-terminal targeting (pre-)sequence. After translocation into the mitochondrial matrix, the presequence is removed by MPP (Brunner, Klaus, and Neupert 1994
). MPP consists of two nonidentical but homologous subunits, designated
-MPP and ß-MPP, which were identified in the mitochondria of several organisms (Braun and Schmitz 1995
). Only the ß subunit has catalytic activity (Arretz et al. 1994
). All known MPP proteins belong to the so-called pitrilysin, or insulinase, family of endoproteases (Barret, Rawlings, and Woesner 1998
). This family includes (1) Escherichia coli pitrilysin, a periplasmic protease involved in the degradation of small peptides; (2) insulinases from mammals and insects; (3) the PqqF protein from Klebsiella, involved in biosynthesis of the coenzyme PQQ; and (4) the N-arginine dibasic convertase from the rat. All catalytically active members of the pitrilysin family contain the motif His-x-x-Glu-His-x76-Glu, in which the two histidine residues and the C-terminal glutamate are required for the binding of zinc (Barret, Rawlings, and Woesner 1998
). This motif is not conserved in
-MPP. An unrooted tree depicting possible evolutionary relationships between known members of the pitrilysin/insulinase family of endoproteases is shown in figure 1
A. It has to be noted, however, that the phylogenetic analysis of this protease family is complicated by the fact that the most distantly related proteins belonging to this family share amino acid sequence similarity only in the regions containing their zinc-binding motifs (data not shown).
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In contrast to mycobacteria and R. prowazekii, B. subtilis is highly amenable to genetic analyses. Thus, the identification of the mlpA gene of B. subtilis offered the exciting possibility to investigate the function of the MlpA protein of this organism in particular, and the evolution of the function of MPP-like proteins in general. For this purpose, the mlpA gene of B. subtilis was disrupted with a kanamycin resistance marker, resulting in a truncation of the MlpA protein at residue 214. Using the mlpA mutant strain (mlpA), it was shown that MlpA is not required for viability, growth, or sporulation. Unexpectedly,
mlpA cells showed about fivefold increased levels of proteolytic activity in their growth medium. As shown with specific inhibitors for serine proteases (PMSF) and metalloproteases (EDTA), this increase concerned the activities of both types of proteases (fig. 2
A), several of which are secreted by B. subtilis (Wong 1995
). In particular, the secretion of subtilisin (AprE), the major secreted serine protease of B. subtilis, was strongly stimulated, whereas the secretion of the neutral protease E (NprE), the major metalloprotease in the medium, was not affected (fig. 2
B). Furthermore, neither the levels of
-amylase nor those of levansucrase in the medium of
mlpA cells were affected (not shown). This suggests that the disruption of mlpA stimulated the expression and/or secretion of a subset of proteins, including AprE and at least one unidentified metalloprotease. To test whether the expression of the aprE gene was stimulated, a transcriptional aprE-lacZ gene fusion was introduced (ectopically) in the chromosome of
mlpA cells and the parental strain. The resulting strains were grown in SSM medium for high production of AprE, and samples, withdrawn at hourly intervals, were assayed for ß-galactosidase activity. As shown in figure 2
C, in the postexponential growth phase, the activity of the aprE promoter was strongly increased in
mlpA cells, showing that the MlpA protein acts as a negative regulator of aprE gene expression. This effect was independent of DegU (data not shown), a key regulator among the nine known regulators of aprE transcription (Smith 1993
). Interestingly, the expression of mlpA itself peaks at the onset of the transcription of the aprE gene (fig. 2
D).
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Footnotes |
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1 Keywords: Bacillus subtilis,
mitochondrial processing peptidase,
gene regulation,
subtilisin.
2 Present address: VTT Biotechnology and Food Research, Espoo, Finland.
3 Present address: Department of Pharmaceutical Biology, University of Groningen, Groningen, the Netherlands
4 Address for correspondence and reprints: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, the Netherlands. E-mail: j.m.van.dijl{at}farm.rug.nl
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