* Department of Pharmaceutical Biology, University of Groningen, Groningen, the Netherlands
Department of Genetics, Smurfit Institute, Trinity College, Dublin, Ireland
Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Haren, the Netherlands
|| Dipartimento di Genetica e Microbiologia, Università degli Studi di Pavia, Pavia, Italy
¶ Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany
# Institute of Biotechnology, ETH Zürich, Zürich, Switzerland
** Génétique Microbienne, INRADomaine de Vilvert, Jouy en Josas, France
Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
Centro de Biología Molecular Severo Ochoa, CSIC, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain
Correspondence: E-mail: j.m.van.dijl{at}farm.rug.nl.
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Abstract |
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Key Words: Bacillus subtilis PBSX prophage secretome skin SPß
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Introduction |
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Characterized genomes of eubacteria differ in size from 580 kb for Mycoplasma genitalium (Fraser et al. 1995) to 9,200 kb for Myxococcus xanthus (He et al. 1994). One of the key questions emerging from the still expanding data set of complete genome sequences is how many genes are essential for the life of an organism such as B. subtilis. Itaya (1995) has used a small set of randomly selected genetic loci in B. subtilis to determine the percentage of genes that could be disrupted without loss of viability. This led to the hypothesis that the minimal B. subtilis genome may comprise about 318562 kb (Itaya 1995) which, given the average size of 1 kb for a bacterial protein-encoding gene, corresponds to 300500 genes (Kunst et al. 1997). In a systematic approach of single gene disruptions covering the complete Bacillus genome, about 270 genes have thus far been found to be indispensable for growth of B. subtilis in a rich medium at 37°C (Kobayashi et al. 2003). This finding is in line with a global transposon mutagenesis study showing that 265350 of the 517 genes of M. genitalium are essential for growth under laboratory conditions (Hutchison et al. 1999). Moreover, it suggests that the majority of the B. subtilis genome would be dispensable for growth under defined conditions. It is intriguing to consider whether such a minimal genome would encode a healthy cell. A first step toward an answer can come from the construction of a Bacillus cell that has been deprived of sequences encoding functions that are nonessential for propagation and fermentation. As a consequence of such a chromosome reduction, cellular metabolite and energy resources would not be expended to maintain and express the deleted genetic information. Thus, the consumption of substrates would be optimally directed toward the synthesis of both essential and desired gene products. Concomitantly, the metabolic waste might decrease, because fewer unwanted proteins are synthesized. In this respect, it is important to note that every dispensable protein produced by a cell factory can represent a potential contaminant in the purification of desired proteinaceous products (Kolisnychenko et al. 2002).
For B. subtilis, a large number of potentially dispensable chromosomal loci can be inferred from the complete nucleotide sequence of the chromosome (4,188-kb) (Kunst et al. 1997). On average, the G + C ratio of the B. subtilis chromosome is 43.5%. However, considerable variation in the GC content can be observed throughout the genome sequence. In particular, 10 relatively large AT-rich islands that represent known prophages (SPß and PBSX) and prophage-like regions are distributed over the chromosome (Zahler et al. 1977; Wood et al. 1990; Takemaru et al. 1995; Kunst et al. 1997). Together with three gene clusters involved in the synthesis of polyketide and peptide antibiotics (the polyketide synthase, fengycin, and surfactin operons), they form a group of possibly dispensable regions (fig. 1). Deletion of these regions would lead to removal of 450 kb of DNA, or 12% of the chromosome. Furthermore, removal of phage and phage-like elements would result in the deletion of a number of autolysins, which can cause a severe problem of cell lysis during industrial fermentations.
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In the present studies, a sequential and cumulative approach was explored to delete large dispensable regions from B. subtilis strain 168. Finally, a multiple deletion strain lacking two prophages (SPß and PBSX), three prophage-like elements (prophage 1, prophage 3, and skin), and the polyketide synthase (pks) operon was constructed. Thus, the genome was reduced by 7.7% or 320 kb. The results show that this genome minimization affects neither cell viability nor the key physiological and developmental processes of B. subtilis.
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Materials and Methods |
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To construct the B. subtilis 6 strain, plasmid-based chromosomal integration-excision systems were used. The strain B. subtilis
SPß (Dorenbos et al. 2002) was used to delete the skin element (26551292699959 bp SubtiList coordinates). To this purpose, the flanking regions of skin were PCR-amplified with the primer pairs YqcM1/YqcM2 and YqaB1/YqaB2, and cloned into the chromosomal integration vector pG+ host4. The resulting plasmid was used to transform B. subtilis
SPß for chromosomal integration into yqcM or yqaB. Transformants (Emr) were selected at 30°C. Next, the excision of the integrated plasmid (together with skin) from the chromosome was provoked by growth at 42°C, resulting in the twofold deleted strain TFC7A (
SPß
skin). PBSX (13129521347172 bp SubtiList coordinates) was deleted from TFC7A using a pG+ host4-based integration-excision plasmid that contains the flanking regions of this prophage, PCR-amplified with the primer pairs YjnA1/YjnA2 and XlyA1/XlyA2. This resulted in strain TF8A (
SPß
skin
PBSX). For deletion of prophage 1 (202092220145 bp SubtiList coordinates) two fragments flanking the prophage 1 region were PCR-amplified with the primer pairs GlmS1/GlmS2 and YbdG1/YbdG2 and ligated into pORI280 after PCR-mediated splicing by overlap extension (Horton et al. 1989). The resulting plasmid, p280GY, was used to transform B. subtilis for chromosomal integration into glmS or ybdG. Transformants (Emr and blue on TY plates with X-gal) were grown in the absence of erythromycin to obtain the fourfold deleted strain
4 (
SPß
skin
PBSX
prophage 1; Ems and white on TY plates with X-gal) due to spontaneous excision of the plasmid from the chromosome (together with the prophage 1 region). The largest part of the pks operon (17813061857233 SubtiList coordinates) in the
4 strain was replaced with a Cmr marker by double cross-over recombination. For this purpose, the pJM105A-based plasmid pJM
80 was used, which contains a pks flanking region amplified with the primer pair EUP1/ELO1 (comprising the 5' end of pksA) and a cloned flanking region of pks (comprising the 5' ends of pksR and pksS). A Cmr marker is located between the two pks flanking regions on pJM
80. This plasmid was first used to replace the pks operon of B. subtilis 168 with the Cmr marker and, subsequently, chromosomal DNA of the resulting strain (PB1862) was used to transform B. subtilis
4. This resulted in the fivefold deleted strain
5 (
SPß
skin
PBSX
prophage 1 pks::cat). The
5 strain was used to delete prophage 3 (651866665067 bp SubtiList coordinates). A derivative of pMTL20E (carrying an Emr marker) was made, which contains two fragments flanking prophage 3 that were PCR-amplified with the primer pairs pre-ydiM1/pre-ydiM2 and post-gutR1/gutR2. A Kmr cassette is located between the two prophage 3 flanking regions. The resulting plasmid was used to transform B. subtilis for chromosomal integration. Transformants (Kmr) were transformed with a pEpUC
1 derivative, which contains the same flanking regions of prophage 3 but lacks the Kmr marker between them. Single cross-over integrants (Kmr Emr) were selected at 51°C. Finally, excision of the integrated pEpUC
1 (together with the Kmr marker) was provoked by growth at 30°C. The resulting Kms Ems strain was named B. subtilis
6 (
SPß
skin
PBSX
prophage 1 pks::cat
prophage 3). The sequential introduction of the deletions was verified by Southern hybridization and/or PCR.
Transcript Profiling
Transcript profiling was performed with Bacillus subtilis Panorama macro-arrays from Sigma-Genosys. Total RNA from B. subtilis 168 was isolated with the High Pure RNA isolation kit of Roche Molecular Biochemicals. For simultaneous reverse transcriptase reactions on all mRNAs in the RNA sample, 4 µg of total RNA was added to 1 pmol of ORF-specific primers (Eurogentec). This mixture was heated to 70°C for 10 min and subsequently stored on ice. Next, 10 µl of 5x first strand buffer (Invitrogen), 5 µl of 0.1 M DTT, 0.5 µl RNasin 40 U/µl (Roche Molecular Biochemicals), and 2.5 µl dNTPs (5 mM dATP, dGTP, dTTP, and 0.1 mM dCTP) were added to the RNA-primer mix. The total reaction volume was adjusted to 42.5 µl with water treated with 0.1% diethyl pyrocarbonate (DEPC). After addition of 5 µl [-33P] dCTP (50 µCi) (Redivue, Amersham Biosciences) and 2.5 µl of Superscript II reverse transcriptase (Invitrogen), the reaction mix was incubated for 2 h at 42°C, followed by 15 min at 70°C. The reaction was stopped and the RNA was denatured by adding 2 µl of 0.5 M EDTA, 2 µl of 10% SDS, and 6 µl of 3 N NaOH. Upon incubation for 30 min at 68°C, the mixture was neutralized by adding 6 µl of 2 N HCl. The [
-33P]-labeled cDNA was purified using Sephadex G-25 columns (Roche Molecular Biochemicals). The percentage of label incorporation was checked by scintillation counting. Prior to hybridization, the hybridization bottles and arrays were washed with 2x SSPE (0.36 M NaCl, 20 mM Na-phosphate buffer pH 7.7, and 2 mM EDTA). The arrays were then pre-hybridized in hybridization solution (Sigma-Genosys) supplemented with 100 µg/ml salmon testis DNA (Sigma-Genosys) for at least 1 h. The labeled cDNA was added to the array after 10 min incubation at 90°95°C in hybridization solution plus salmon testis DNA. Hybridization was performed for 1218 h. After it was washed with washing solution (0.5x SSPE, 0.2% SDS), the array was wrapped in Saran wrap and exposed to phosphoimager screens (Packard Instrument Company) for 2 or 3 days. The screens were scanned by the Cyclone Imager (Packard Instrument Company). The Array-Pro Analyzer 4.0 software package (Media Cybernetics) was used to analyze the images. The signals from duplicate spots were averaged and the intensities were expressed as percentages of the total signal.
Metabolic Flux Ratio (METAFoR) Analysis
Biomass aliquots of B. subtilis were harvested during late exponential growth at OD600 nm values between 2.5 and 3.0. Biomass pellets from 2 ml of culture broth were washed once with 1 ml 0.9% (w/v) NaCl and hydrolyzed in 1.5 ml 6 M HCl at 110°C for 24 h in sealed glass tubes. The dried hydrolysate was derivatized with N-(tert-butyldimethylsilyl)-N-methyl-trifluoracetamide (Fluka) and subjected to GC-MS analysis as described previously (Dauner and Sauer 2000). The GC-MSderived mass distributions in proteinogenic amino acids were then used to calculate intracellular carbon flux ratios using probabilistic equations (Fischer and Sauer 2003) and a metabolic network model for B. subtilis (Sauer et al. 1996). The metabolic by-products acetate and acetoin were determined by high-performance liquid chromatography (HPLC). Glucose concentrations were measured with the Beckman Synchron CX5CE autoanalyzer using the glucose reagent kit supplied by Beckman. Maximum growth rates (µmax) were determined by log-linear regression analysis of OD600 nm versus time, with µmax as the regression coefficient. Dry matter concentrations of biomass were calculated using a predetermined correlation factor of 0.33 g cellular dry weight per OD600 nm unit.
Competence, Sporulation, and Spore Germination Assays
The B. subtilis 6 and the parental 168 strains were tested for competence using the two-step method as described previously (Bron and Venema 1972). The strains were transformed with chromosomal DNA of B. subtilis OG1 (trp+), and transformants were selected for tryptophane prototrophy on minimal agar without tryptophane. Transformability was expressed as the number of transformants relative to the total viable count. The ability of the strains to sporulate and germinate was tested by growing 25 colonies for 2 to 3 days on a sporulation medium agar plate at room temperature. The colonies were transferred onto a filter paper, after which they were exposed to chloroform vapor in a vacuum chamber for 45 min. The filters were used to make a contact replica on TY plates, which were incubated overnight at 37°C. Finally, the number of colonies growing on the replica plates was counted. Sporulation was also tested by growing the cells overnight in sporulation medium, after which an aliquot of the culture was heated to 80°C for 10 min. Subsequently, the presence of viable spores was assayed by plating.
Proteomics
The B. subtilis 6 mutant strain and the parental strain 168 were grown at 37°C under vigorous agitation in 1 liter TY medium. After 1 h of post-exponential growth, cells were separated from the growth medium by centrifugation. The secreted proteins in the growth medium were collected for two-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), as previously described (Jongbloed et al. 2002; Antelmann et al. 2001). Two-dimensional gel image analysis was performed with the Decodon Delta 2D software, which is based on dual channel image analysis (Bernhardt et al. 1999). Using this software, the master image (represented by green protein spots) is warped with the sample image (represented by red protein spots) after specific vector points have been set. Consequently, green protein spots in the dual channel image are predominantly present in the master image, whereas red protein spots are predominantly present in the sample image. Yellow protein spots are present at similar amounts in both images. After background subtraction, normalization is performed in order to equalize the gray values in each image. Each experiment was repeated at least two times.
Enzyme Activity Assays
To determine lipase (i.e., esterase) activity, the colorimetric assay as described by Lesuisse, Schanck, and Colson (1993) was applied with some modifications. In short, 180 µl of reaction buffer (0.1 M H2KPO4 pH 8.0, 0.1% Arabic gum, 0.36% Triton X-100) was supplemented with 10 µl of the chromophoric ligand 4-nitrophenyl caprylate (10 mM in methanol). The reaction was started by the addition of 10 µl of culture supernatant. Lipase activity was determined by measuring the increase in absorbance at 405 nm/min of incubation at room temperature, per OD600 of the culture at the time of sampling.
Protease activity was quantified with azocasein (Sigma). Growth medium (250 ml) was mixed with 2% azocasein suspension (150 ml) in 50 mM Tris-HCl (pH 7.5), 4 mM CaCl2, and incubated for 60 min at 25°C. The reaction was stopped by the addition of 1.2 ml 10% TCA. After centrifugation, absorbance changes (440 nm) of the supernatant were determined.
-Amylase activity was assayed with a halo assay. Cells were grown to the stationary phase, after which they were separated from the growth medium by centrifugation. The medium fractions were spotted on Durapore membrane filters (Millipore) that were placed on TY-agar plates containing 1% starch (Merck). The amounts of medium spotted on the filters were corrected for the OD600 nm of each culture. After overnight incubation at 37°C, the plates were analyzed for starch degradation by staining with iodine vapor. Diameters of the resulting clear zones (halos) were measured.
To assay ß-galactosidase activity, overnight cultures were diluted in fresh medium and samples were taken at different time intervals for OD600 nm readings and ß-galactosidase activity determinations. For strains containing a transcriptional lacZ fusion, the ß-galactosidase assay and the calculation of ß-galactosidase units (Miller units: nmol.OD6001·min1) were performed as described by Hyyryläinen et al. (2001). Experiments were repeated at least twice, starting from independently obtained transformants. In all experiments, the relevant controls were performed in parallel. Although some differences were observed in the absolute ß-galactosidase activities, the ratios between these activities in the various strains tested were largely constant.
Western Blotting and Immunodetection
To assay the B. amyloliquefaciens -amylase (AmyQ) production levels, cells were separated from the growth medium by centrifugation. Samples for SDS-PAGE were prepared as described previously (van Dijl et al. 1991). After separation by SDS-PAGE, proteins were transferred to a Protran nitrocellulose transfer membrane (Schleicher and Schuell) as described by Kyhse-Andersen (1984). AmyQ and LipA were visualized with specific antibodies and horseradish peroxidase- or alkaline phosphatase-anti-rabbit IgG conjugates (Jackson ImmunoResearch).
Motility Plate Assay
To study the motility of Bacillus cells, 2 µl aliquots of overnight cultures were transferred to a TY-agarose plate containing 0.75%, 0.5%, or 0.27% agarose. Before transfer to the plates, the OD600 nm of each culture was measured and adjusted to 1. After overnight incubation at 37°C, the degree of swarming was assessed.
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Results |
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Six functional categories have been assigned to the genes of B. subtilis (Kunst et al. 1997; Moszer 1998). To take an inventory of the genes that have been removed from the 168 strain, we compared the functional categories of the deleted genes (332 in total) with the functional classification of all B. subtilis protein-encoding genes (Kunst et al. 1997; Moszer 1998) (table 2). Compared to the average in the chromosome, the percentage of genes that specify proteins with no similarity to other proteins (functional category number 6) is much higher among the deleted genes. As was to be expected, most of the deleted genes within the "other functions category" (number 4) encode proteins with phage-related functions. To gain insight into the actual gene functions that are absent from the 6 strain, a search for deleted genes encoding proteins with a known function or showing similarity to known proteins (functional categories 14) was performed using the SubtiList database (http://genolist.pasteur.fr/SubtiList/) (Kunst et al. 1997; Moszer 1998). The results are documented in table 3. With respect to "health" of B. subtilis
6, it is interesting to note that this strain lacks 12 genes which have been implicated in cell lysis. Furthermore, SPß encodes the lantibiotic sublancin 168 (SunA). Deletion of SPß leads to sublancin 168 sensitivity (Dorenbos et al. 2002).
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The skin (for sigK intervening) element is positioned within the sigK gene. It is excised at a particular stage of sporulation, leading to the reconstitution of sigK by fusion of the spoIIIC and spoIVCB genes. The sigma factor K, encoded by sigK, is required for sporulation (Errington et al. 1988; Kunkel et al. 1988; Stragier et al. 1989). In our mutant, the skin element has been deleted from arsC to yqaB, leaving the spoIIIC and spoIVCB genes intact. Furthermore, skin contains the phrE/rapE genes which, like the phrA/rapA genes from PBSX, are involved in the early stages of the sporulation process (Lazazzera et al. 1999; Perego 1999). To investigate the effect of the six combined deletions on sporulation, we analyzed spore formation and germination in B. subtilis
6. None of these developmental processes was detectably influenced in this strain (data not shown). This demonstrates that the combined deletion of known determinants for sporulation encoded by skin and PBSX does not affect sporulation.
Changes in the Extracellular Proteome
To investigate the effects of the large deletions on protein secretion in general, the extracellular proteomes of B. subtilis 6 and 168 were compared by employing 2D PAGE-MS. A representative result is shown in figure 3A, in which dual channel imaging was used to monitor possible changes in extracellular protein composition. Specifically, the phage proteins YolA (SPß), XlyA, XkdG, XkdK, and XkdM (PBSX) were shown to be absent from the growth medium of B. subtilis
6 (represented as green protein spots in fig. 3A). Furthermore, the amounts of the lipoproteins MntA (manganese transport) and YfiY (possibly involved in iron(III) dicitrate transport) are reproducibly detected at increased levels in the medium of the B. subtilis
6 strain (red protein spots), whereas the secreted esterase LipA was present in strongly decreased amounts (green protein spot). The latter observation was remarkable, as the lipA gene is still present in B. subtilis
6. To verify whether the decrease in the secretion of LipA by B. subtilis
6 is significant, this strain was transformed with pLip2031. The presence of this plasmid results in the overproduction of LipA. Next, LipA secretion was monitored both by activity assays and by Western blotting. The results showed that, under overproducing conditions, the secretion of LipA by B. subtilis
6 is reduced about threefold as compared to the parental strain 168 (data not shown).
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High-Level Secretion of Active AmyQ by B. subtilis 6
To assess the secretion capacity of the 6 strain, we studied the secretion of active AmyQ of Bacillus amyloliquefaciens with an
-amylase plate assay. For this purpose, the 168 and
6 strains were transformed with plasmid pKTH10, which results in the high-level production and secretion of AmyQ (Palva 1982). The results demonstrated that the deletion strain can still secrete active AmyQ, a heterologous protein (data not shown). Importantly, B. subtilis
6 pKTH10 did not reveal a significant difference in secretion of AmyQ compared to the parental strain containing pKTH10. Likewise, the secretion of the endogenous
-amylase AmyE by the
6 strain was not affected, which is consistent with the 2D gel analysis (fig. 3A; yellow AmyE spot).
Previous studies have shown that the high-level production of AmyQ imposes a so-called secretion stress on cells of B. subtilis. This stress is sensed by the CssRS two-component regulatory system. Activation of the CssRS system results in the transcription of the htrA and htrB genes at significantly elevated levels (Hyyryläinen et al. 2001; Darmon et al. 2002). Consequently, the level of htrA or htrB transcription can be used as an indicator for secretion stress. To study the induction of secretion stress by AmyQ in B. subtilis 6 pKTH10, an htrB-lacZ transcriptional gene fusion was introduced in this strain. Next, ß-galactosidase activities in cells grown in TY medium at 37°C were measured as a function of time. As shown in figure 4A (closed symbols), the transcription of htrB-lacZ was not affected by the
6 mutations, demonstrating that these mutations do not trigger a secretion stress response. Moreover, B. subtilis
6 pKTH10 containing the htrB-lacZ fusion displayed an AmyQ-induced secretion stress response that is very similar to that of the parental strain transformed with pKTH10 (fig. 4A, open symbols). Consistent with this observation, Western blotting analyses revealed that the AmyQ-producing cells of the
6 strain and the parental strain 168 contained comparable amounts of the precursor and mature forms of this secretory protein (fig. 4B). Furthermore, similar amounts of mature AmyQ protein were detectable in the growth medium, which is in accordance with the results of the activity assay that was performed (fig. 4B and data not shown). Proteomics was employed to monitor possible effects of AmyQ-induced secretion stress on the composition of the extracellular proteome of B. subtilis
6. The dual-channel analysis of 2D gels (fig. 3B) revealed that the overproduction of AmyQ has only a few significant consequences for the composition of the extracellular proteome of the
6 strain. First, a number of AmyQ spots with a slightly different pI is detectable in the medium of B. subtilis
6 pKTH10. Furthermore, the extracellular level of HtrA, a known indicator of the AmyQ-induced secretion stress response, is significantly increased in the
6-derived sample. Unexpectedly, the high-level production of AmyQ in the
6 strain also results in the extracellular appearance of the PtsH (or HPr) protein, a histidine-containing phosphocarrier protein of the phosphotransferase system. Taken together, these observations show that the absence of 332 genes from B. subtilis
6 has no major impact on protein secretion in general, or on the high-level secretion of the heterologous protein AmyQ in particular.
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Discussion |
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A normal maintenance and replication of the B. subtilis chromosome is required for successful progress through the developmental processes of competence and sporulation (Piggot and Coote 1976; Ireton and Grossman 1992; Ireton, Gunther, and Grossman 1994; Sciochetti, Piggot, and Blakely 2001). Because these processes are not disturbed in B. subtilis 6, it can be concluded that the over all chromosome "physiology" (i.e., the sum of all processes required for chromosome replication and maintenance) is not significantly disturbed by the combined deletions. Nevertheless, it remains to be determined whether specific processes, such as chromosome condensation and packaging and global distribution of transcriptional signals are completely unaffected. On the one hand, the apparent lack of effect on chromosome physiology is not surprising, because the deletions are almost evenly distributed along the chromosome:
141 kb (3.3%) has been deleted from the 0172° replichore and
180 kb (4.3%) from the 172360° replichore (fig. 1). On the other hand, competence development was seemingly not affected in the intermediate strains, in which the relative sizes of the replichores are more affected (unpublished observations). The view that the stepwise deletions created in the present studies do not interfere with chromosome physiology would be in line with a stepwise invasion of prophage(-like) regions during evolution of the B. subtilis chromosome.
The present transcript-profiling experiments with DNA arrays suggest that genes in the prophage(-like) regions are expressed at a relatively lower level than genes in the non-prophage regions. Although the efficiency of the DNA hybridizations in these experiments may be somewhat biased by the fact that prophage genes have a higher AT content than the remaining genes, the results are likely to be biologically relevant, because the prophages SPß and PBSX are not induced under standard laboratory conditions. Although genes of these prophages are most likely repressed, at least two other explanations for the low transcription levels of prophage genes and genes in prophage-like regions are conceivable. First, the low transcription of most of these genes may relate to their presumed foreign origin. For example, promoters of prophage(-like) genes may be optimal for other organisms, which can result in a poor match with authentic promoter sequences of B. subtilis. Second, several of these genes belong to the 25% of B. subtilis genes that are transcribed in the opposite direction of replication fork movement (Kunst et al. 1997; Rocha et al. 2000). This may interfere with their efficient transcription. It has to be noted that, for unknown reasons, the relative transcription levels of prophage 1 and prophage 6 genes are comparable with those of the non-prophage genes of B. subtilis.
At present it is not clear to what extent the 6 strain represents an improved bacterial cell factory. Clearly, the capacity for high-level production and secretion of a heterologous model protein was neither positively nor negatively affected by the mutations in this strain. This suggests that no large energy resources were redirected toward product formation. Furthermore, only a few proteins were absent from the extracellular proteome, indicating that the
6 strain is only marginally improved in terms of the removal of unwanted by-products. It is presently not clear why the amounts of MntA and YfiY were significantly increased in the medium of B. subtilis
6, while the extracellular accumulation of LipA was reduced. Furthermore, no major changes were observed in the cellular proteome of B. subtilis
6, which is consistent with the fact that no prophage proteins are detectable on the cellular proteome of the parental strain (H. Antelmann and M. Hecker; unpublished data). In fact, these findings are consistent with the low transcriptional levels of the prophage-like genes. Importantly, the analysis of the extracellular proteome of B. subtilis
6 revealed that the sensitivity of this strain to cell lysis is not increased, even under conditions of severe secretion stress. The only major changes observed under these conditions concerned the presence of increased amounts of HtrA and PtsH in the medium. The increased level of extracellular HtrA is consistent with the induction of htrA transcription under conditions of secretion stress (Hyyryläinen et al. 2001). At present it is not known whether the elevated levels of PtsH reflect a physiological response of the
6 strain to protein secretion stress.
Thus far, the 6 strain seems to be moderately inferior to the parental strain 168 in only two aspects. First, B. subtilis
6 lacks the gene for the thiol-disulfide oxidoreductase BdbB, which plays a minor role in the heterologous secretion of the E. coli PhoA protein by B. subtilis (Bolhuis et al. 1999). Probably, BdbB supports its paralog BdbC (which is present in B. subtilis
6) in the formation of two disulfide bonds in PhoA. Second, together with the deletion of the prophages, the respective immunity regions were lost. If necessary for production purposes, the bdbB gene as well as the phage immunity regions can be re-inserted into the chromosome of B. subtilis
6.
In terms of cell factory engineering, B. subtilis 6 has one major advantage over conventional B. subtilis production strains: it lacks the BsuM restriction-modification system. As shown by Ohshima et al. (2002), prophage 3 encodes both the genes for BsuM modification (ydiO and ydiP) and BsuM restriction (ydiR, ydiS, and ydjA). BsuM restriction has been shown to reduce the transformation efficiency of B. subtilis with recombinant plasmids up to 7,800-fold (Haima, Bron, and Venema 1987). Moreover, this system was found to be responsible for structural plasmid instability in B. subtilis, which limits the application potential of plasmids for high-level protein production. With respect to bacterial evolution for survival in the soil, it is interesting to speculate that B. subtilis has recruited prophage 3, specifying the BsuM system, in order to protect itself against invading foreign DNA.
Finally, it was recently shown that 12 K-islands could be deleted from the E. coli genome, resulting in an 8.1% reduced genome size (Kolisnychenko et al. 2002). Unfortunately, apart from growth experiments, which revealed no major differences with the parental strain MG1655, a detailed phenotypical analysis has so far not been documented for this engineered E. coli strain. In the present studies, we have taken the phenotypic characterization of the first B. subtilis strain with a reduced genome a few steps further by a combination of transcript profiling, metabolic flux analysis, proteomics, and dedicated assays for competence development, sporulation, protein secretion, and cell motility. We are convinced that such an integrated approach will be of utmost importance for the functional genomic characterization of organisms such as E. coli and B. subtilis. In particular, a combination of genome reduction, transcript profiling, proteomics, and metabolomics seems necessary to attribute physiological functions to the large families of paralogous genes in these organisms. This will also lead to optimized "next generation cell factories." The deletion of two gene families with 3 paralogs, 26 families with 2 paralogs, and 227 unique genes represents an important step toward achieving these goals for B. subtilis.
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Acknowledgements |
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Footnotes |
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Literature Cited |
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