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ABSTRACT: Engineering of Rap-Phr quorum-sensing systems of Bacillus subtilis and subsequent evaluation of the transcription of the aprE gene, encoding a major extracellular alkaline protease.
Addition of synthetic Phr pentapeptides to the growth medium, or overproduction of pre-Phr peptides, slightly improved the transcription of the aprE gene in B. subtilis. Disruption of certain rap genes similarly improved the transcription of the aprE gene. The production of extracellular proteolytic enzymes was increased when the rapA mutation was combined with a degU32 (Hy) mutation for hyper-secretion.
Certain Rap-Phr systems of B. subtilis seem to suppress extracellular AprE production. Although this may be an important feature under natural conditions, repression of AprE production by these systems is not desirable under fermentation conditions.
Although the levels of aprE transcriptional increase in this study are moderate, engineering of Rap-Phr systems may be used to improve the yield of Bacillus strains that are used for the production of the extracellular protease AprE, or Bacillus strains that use of the aprE promoter for the production of a heterologous protein.
Journal of Applied Microbiology 02/2004; 96(3):569-78. · 2.34 Impact Factor
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ABSTRACT: Overproduced alpha-amylases in Bacillus subtilis provoke a specific stress response involving the CssRS two-component system, which controls expression of the HtrA and HtrB proteases. Previously, the B. subtilis TepA protein was implicated in high-level alpha-amylase secretion. Our present studies were aimed at investigating a possible role of TepA in secretion stress management, and characterizing the intensity of the secretion stress response in relation to alpha-amylase production.
The expression of a transcriptional htrB-lacZ gene fusion, and the levels of alpha-amylase production were monitored simultaneously using tepA mutant B. subtilis strains. TepA was shown to be dispensable for secretion stress management. Importantly, however, the levels of htrB-lacZ expression can be correlated with the levels of alpha-amylase production.
Our observations show that the secretion stress response can serve as an indicator for alpha-amylase production levels.
Conceivably, this stress response can be employed to monitor the biotechnological production of various secretory proteins by the Bacillus cell factory.
Letters in Applied Microbiology 02/2004; 39(1):65-73. · 1.62 Impact Factor
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K Kobayashi,
S D Ehrlich,
A Albertini,
G Amati,
K K Andersen,
M Arnaud,
K Asai,
S Ashikaga,
S Aymerich,
P Bessieres, [......],
A Wipat,
H Yamamoto,
M Yamamoto,
Y Yamamoto,
K Yamane,
K Yata,
K Yoshida,
H Yoshikawa,
U Zuber,
N Ogasawara
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ABSTRACT: To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.
Proceedings of the National Academy of Sciences 05/2003; 100(8):4678-83. · 9.68 Impact Factor
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ABSTRACT: Chromosomes of several Bacillus subtilis strains were shown to contain homologs of the ISBsu2 mobile genetic element, which was earlier revealed in a cryptic plasmid of a soil strain of B. subtilis.
Genetika 01/2003; 38(12):1719-22. · 0.44 Impact Factor
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ABSTRACT: A cryptic plasmid from a soil strain of Bacillus subtiliswas found to contain a sequence having features of an IS element. Homologous sequences were also found in the chromosome of this strain and in the chromosomes of some other B. subtilis strains.
Microbiology 12/2002; 72(1):58-63. · 3.06 Impact Factor
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ABSTRACT: Cellular lysis properties of strains of Bacillus subtilis deficient in the synthesis of extracellular proteases was investigated. In all cases, extracellular protease deficiency was found to increase the extent of cellular lysis of batch cultured strains following the transition to stationary phase, the time at which extracellular degradative enzymes are secreted in large quantities. The data indicates that the major extracellular proteases, NprE and AprE, are primarily responsible for the control of this autolytic activity in B. subtilis and has implications for the use of extracellular protease-deficient strains as hosts for the production of heterologous proteins.
Letters in Applied Microbiology 01/2002; 29(2):141 - 145. · 1.62 Impact Factor
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ABSTRACT: The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms. Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification. For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis. In the present studies, proteomic approaches were used to define the extracellular complement of the B. subtilis secretome. Using different growth conditions and a hyper-secreting mutant, approximately 200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry. These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal. Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19). To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B. subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed. Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate. Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium. In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for approximately 50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.
Genome Research 10/2001; 11(9):1484-502. · 13.61 Impact Factor
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H L Hyyryläinen,
A Bolhuis,
E Darmon,
L Muukkonen,
P Koski,
M Vitikainen,
M Sarvas,
Z Prágai, S Bron,
J M van Dijl,
V P Kontinen
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ABSTRACT: The Gram-positive eubacterium Bacillus subtilis is well known for its high capacity to secrete proteins into the environment. Even though high-level secretion of proteins is an efficient process, it imposes stress on the cell. The present studies were aimed at the identification of systems required to combat this so-called secretion stress. A two-component regulatory system, named CssR-CssS, was identified, which bears resemblance to the CpxR-CpxA system of Escherichia coli. The results show that the CssR/S system is required for the cell to survive the severe secretion stress caused by a combination of high-level production of the alpha-amylase AmyQ and reduced levels of the extracytoplasmic folding factor PrsA. As shown with a prsA3 mutation, the Css system is required to degrade misfolded exported proteins at the membrane-cell wall interface. This view is supported by the observation that transcription of the htrA gene, encoding a predicted membrane-bound protease of B. subtilis, is strictly controlled by CssS. Notably, CssS represents the first identified sensor for extracytoplasmic protein misfolding in a Gram-positive eubacterium. In conclusion, the results show that quality control systems for extracytoplasmic protein folding are not exclusively present in the periplasm of Gram-negative eubacteria, but also in the Gram-positive cell envelope.
Molecular Microbiology 10/2001; 41(5):1159-72. · 5.01 Impact Factor
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ABSTRACT: Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.
Journal of Bacteriology 09/2001; 183(16):4752-60. · 3.83 Impact Factor
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ABSTRACT: The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability. As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential. This allows a distinction between SPases that are of major and minor importance for cell viability. Notably, the functional difference between major and minor SPases is not reflected clearly in sequence alignments. Here, we have successfully used molecular phylogeny to predict major and minor SPases. The results were verified with SPases from various bacilli. As predicted, the latter enzymes behaved as major or minor SPases when expressed in B. subtilis. Strikingly, molecular modeling indicated that the active site geometry is not a critical parameter for the classification of major and minor Bacillus SPases. Even though the substrate binding site of the minor SPase SipV is smaller than that of other known SPases, SipV could be converted into a major SPase without changing this site. Instead, replacement of amino-terminal residues of SipV with corresponding residues of the major SPase SipS was sufficient for conversion of SipV into a major SPase. This suggests that differences between major and minor SPases are based on activities other than substrate cleavage site selection.
Journal of Biological Chemistry 08/2001; 276(27):25230-5. · 4.77 Impact Factor
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ABSTRACT: When Bacillus subtilis is subjected to phosphate starvation, genes of the Pho regulon are either induced or repressed. Among those induced are genes encoding alkaline phosphatases (APases). A set of isogenic mutants, with a beta-galactosidase gene transcriptionally fused to the inactivated target gene, was used to identify genes that influence the operation of the Pho regulon. One such gene was nhaC (previously yheL). In the absence of NhaC, growth and APase production were enhanced, while the production of other non-Pho-regulon secretory proteins (proteases and alpha-amylase) did not change. The influence of NhaC on growth, APase synthesis, and its own expression was dependent on the external Na+ concentration. Other monovalent cations such as Li+ or K+ had no effect. We propose a role for NhaC in the uptake of Na+. nhaC appears to be encoded by a monocistronic operon and, contrary to previous reports, is not in the same transcriptional unit as yheK, the gene immediately upstream. The increase in APase production was dependent on an active PhoR, the sensor kinase of the two-component system primarily responsible for controlling the Pho regulon. Transcriptional fusions showed that the phoPR operon and both phoA (encoding APaseA) and phoB (encoding APaseB) were hyperinduced in the absence of NhaC and repressed when this protein was overproduced. This suggests that NhaC effects APase production via phoPR.
Journal of Bacteriology 05/2001; 183(8):2505-15. · 3.83 Impact Factor
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ABSTRACT: We have delineated the amino acid to nucleotide contacts made by two interacting dimers of the replication terminator protein (RTP) of Bacillus subtilis with a novel naturally occurring bipolar replication terminus by converting RTP to a site-directed chemical nuclease and mapping its cleavage sites on the terminus. The data show a relatively symmetrical arrangement of the amino acid to base contacts, and a comparison of the bipolar contacts with that of a normal unipolar terminus suggests that the DNA-protein contacts play an important determinative role in generating polarity from structurally symmetrical RTP dimers. The amino acid to nucleotide contacts provided distance constraints that enabled us to build a three-dimensional model of the protein-DNA complex. The model is consistent with features of the bipolar Ter.RTP complex derived from mutational and cross-linking data. The bipolar terminus arrested Escherichia coli DNA replication and DnaB helicase and T7 RNA polymerase in vitro in both orientations. RTP arrested the unwinding of duplex DNA on the bipolar Ter DNA substrate regardless of the length of the duplex DNA. The latter result suggested further that the terminus arrested authentic DNA unwinding by the helicase rather than just translocation of helicase on DNA.
Journal of Biological Chemistry 05/2001; 276(16):13160-8. · 4.77 Impact Factor
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ABSTRACT: The Gram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory pre-proteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are structurally different from that enzyme, being almost half the size and containing one membrane anchor instead of two. To investigate whether the unique membrane anchor of Bacillus SPases is required for in vitro activity, soluble forms of SipS of B. subtilis, SipS of Bacillus amyloliquefaciens and SipC of the thermophile Bacillus caldolyticus were constructed. Of these three proteins, only a hexa-histidine-tagged soluble form of SipS of B. amyloliquefaciens could be isolated in significant quantities. This protein displayed optimal activity at pH 10, which is remarkable considering the fact that the catalytic domain of SPases is located in an acidic environment at the outer surface of the membrane of living cells. Strikingly, in contrast to what has been previously reported for the soluble form of the E. coli SPase, soluble SipS was active in the absence of added detergents. This observation can be explained by the fact that a highly hydrophobic surface domain of the E. coli SPase, implicated in detergent-binding, is absent from SipS.
Microbiology 05/2001; 147(Pt 4):909-17. · 3.06 Impact Factor
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ABSTRACT: The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.
Journal of Biological Chemistry 01/2001; 275(52):41350-7. · 4.77 Impact Factor
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ABSTRACT: Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of Escherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.
Journal of Bacteriology 11/2000; 182(20):5765-70. · 3.83 Impact Factor
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ABSTRACT: One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.
Microbiology and Molecular Biology Reviews 10/2000; 64(3):515-47. · 13.02 Impact Factor
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ABSTRACT: Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalent, but structurally quite different, forming two distinct SPase families that share only few conserved residues. P-type SPases were, so far, exclusively identified in eubacteria and organelles, whereas ER-type SPases were found in the three kingdoms of life. Strikingly, the presence of ER-type SPases appears to be limited to sporulating Gram-positive eubacteria. The present studies were aimed at the identification of potential active site residues of the ER-type SPase SipW of Bacillus subtilis, which is required for processing of the spore-associated protein TasA. Conserved serine, histidine, and aspartic acid residues are critical for SipW activity, suggesting that the ER-type SPases employ a Ser-His-Asp catalytic triad or, alternatively, a Ser-His catalytic dyad. In contrast, the P-type SPases employ a Ser-Lys catalytic dyad (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). Notably, catalytic activity of SipW was not only essential for pre-TasA processing, but also for the incorporation of mature TasA into spores.
Journal of Biological Chemistry 09/2000; 275(33):25102-8. · 4.77 Impact Factor
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Molecular Biology and Evolution 02/2000; 17(1):198-201. · 5.55 Impact Factor
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ABSTRACT: The growth and protein export defects of Escherichia coli secA51(Ts) strains can be suppressed by the CsaA protein of Bacillus subtilis. The present studies indicate that this effect can be attributed to chaperone-like activities of CsaA. First, CsaA stimulated protein export in secB, groES and dnaJ mutant strains of E. coli. Second, CsaA suppressed the growth defects of dnaK, dnaJ and grpE mutants of E. coli. Third, and most importantly, CsaA exhibited chaperone-like properties by stimulating the reactivation of heat-denatured firefly luciferase in groEL, groES, dnaK and grpE mutant strains of E. coli, and by preventing the aggregation of heat-denatured luciferase in vitro. Thus, it seems that CsaA suppresses the growth and secretion defects of E. coli secA(Ts) strains either by improving the translocation competence of exported pre-proteins, thereby making them better substrates for mutant SecA proteins, or by stimulating the translocation activity of mutant SecA proteins.
Microbiology 02/2000; 146 ( Pt 1):77-88. · 3.06 Impact Factor
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ABSTRACT: Type II signal peptidases (SPase II) remove signal peptides from lipid-modified preproteins of eubacteria. As the catalytic mechanism employed by type II SPases was not known, the present studies were aimed at the identification of their potential active site residues. Comparison of the deduced amino acid sequences of 19 known type II SPases revealed the presence of five conserved domains. The importance of the 15 best conserved residues in these domains was investigated using the type II SPase of Bacillus subtilis, which, unlike SPase II of Escherichia coli, is not essential for viability. The results showed that only six residues are important for SPase II activity. These are Asp-14, Asn-99, Asp-102, Asn-126, Ala-128, and Asp-129. Only Asp-14 was required for stability of SPase II, indicating that the other five residues are required for catalysis, the active site geometry, or the specific recognition of lipid-modified preproteins. As Asp-102 and Asp-129 are the only residues invoked in the known catalytic mechanisms of proteases, we hypothesize that these two residues are directly involved in SPase II-mediated catalysis. This implies that type II SPases belong to a novel family of aspartic proteases.
Journal of Biological Chemistry 11/1999; 274(40):28191-7. · 4.77 Impact Factor
