Global Gene Expression Profiling of Bacillus subtilis ...

J Mol Microbiol Biotechnol 2007;12:121–130 DOI: 10.1159/000096467

Global Gene Expression Profiling of Bacillus subtilis in Response to Ammonium and Tryptophan Starvation as Revealed by Transcriptome and Proteome Analysis Le Thi Tam Christine Eymann Haike Antelmann Dirk Albrecht Michael Hecker Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany

Key Words Bacillus subtilis Proteome Transcriptome Ammonium starvation Tryptophan starvation CcpA

Abstract The global gene expression profile of Bacillus subtilis in response to ammonium and tryptophan starvation was analyzed using transcriptomics and proteomics which gained novel insights into these starvation responses. The results demonstrate that both starvation conditions induce specific, overlapping and general starvation responses. The TnrA regulon, the glutamine synthetase ( glnA) as well as the Ldependent bkd and roc operons were most strongly and specifically induced after ammonium starvation. These are involved in the uptake and utilization of ammonium and alternative nitrogen sources such as amino acids, -aminobutyrate, nitrate/nitrite, uric acid/urea and oligopeptides. In addition, several carbon catabolite-controlled genes (e.g. acsA, citB), the -acetolactate synthase/-decarboxylase alsSD operon and several aminotransferase genes were specifically induced after ammonium starvation. The induction of F- and E-dependent sporulation proteins at later time points in ammonium-starved cells was accompanied by an Web access: Tables 1 and 2 are available as supplemental material online (http://microbio1.biologie.uni-greifswald.de/publications. html).

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increased sporulation frequency. The specific response to tryptophan starvation includes the TRAP-regulated tryptophan biosynthesis genes, some RelA-dependent genes (e.g. adeC, ald ) as well as spo0E. Furthermore, we recognized overlapping responses between ammonium and tryptophan starvation (e.g. dat, maeN ) as well as the common induction of the CodY and H general starvation regulons and the RelA-dependent stringent response. Many genes encoding proteins of so far unknown functions could be assigned to specifically or commonly induced genes. Copyright © 2007 S. Karger AG, Basel

Introduction

In natural ecosystems, bacteria are subjected to a variety of starvation conditions and have therefore developed a highly sophisticated network of adaptational responses to cope with these growth-restricting situations. Important strategies for the adaptation to nutrient depletion in Bacillus subtilis are the synthesis of degradative enzymes, the development of genetic competence [Sonenshein, 1989], the stringent response [Cashel et al., 1996], the B-dependent general stress response [Hecker and Völker, 2001; Price, 2002] and the sporulation process [Hoch, 1993]. Bacteria are able to monitor the availability of essential nutrients (carbon, nitrogen, phosphorus) and to transmit this information to regulatory pro-

Christine Eymann Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald F.-L.-Jahn-Strasse 15, DE–17487 Greifswald (Germany) Tel. +49 3834 864 227, Fax +49 3834 864 202 E-Mail [email protected]

teins. Furthermore, bacteria preferentially utilize those carbon or nitrogen sources which can be metabolized most rapidly [Fisher and Sonenshein, 1991]. In B. subtilis, glutamine is the preferred nitrogen source followed by arginine and ammonium. In contrast to enteric bacteria, B. subtilis lacks an assimilatory glutamate dehydrogenase. Thus, ammonium assimilation occurs via the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway only. The expression of proteins involved in nitrogen metabolism is controlled by TnrA, GlnR and CodY in response to the availability of nitrogen sources [Fisher and Débarbouillé, 2002]. TnrA functions as activator and repressor of transcription under nitrogen-restricted conditions. Most of the positively regulated TnrA target genes might be involved in the utilization of ammonium (nrgAB), glutamine (glnQHMP) and alternate nitrogen sources, such as asparagine (asnZ), -aminobutyrate (gabP), nitrate and nitrite (nas), alanine (yrbD), uric acid (puc), urea (ureABC) and oligopeptides (ykfD, opp) [Fisher and Débarbouillé, 2002; Yoshida et al., 2003]. TnrA represses the transcription of glnRA (glutamine synthetase) and gltAB (glutamate synthase) under conditions of nitrogen limitation. In contrast, GlnR acts as repressor of glnRA, ureABC and tnrA in cells grown with nitrogen excess [Fisher and Débarbouillé, 2002]. The glutamine synthetase protein (GlnA) is required for the transduction of a nitrogen signal to GlnR and TnrA [Schreier and Sonenshein, 1986; Wray et al., 1996]. Under nitrogen excess conditions the feedback-inhibited GlnA protein binds to TnrA and blocks the DNA-binding activity of TnrA [Wray et al., 2001]. CodY, a third regulatory protein, represses the transcription of several genes involved in nitrogen and carbon metabolism as well as in competence, sporulation and motility during the fast growth in the presence of nutrient excess [Serror and Sonenshein, 1996; Molle et al., 2003]. Amino acid starvation triggers the stringent response in B. subtilis – a more general response to amino acid or nitrogen starvation [Wendrich and Marahiel, 1997; Eymann et al., 2002]. Tryptophan starvation leads to the transcriptional activation of the trp operon encoding tryptophan biosynthetic enzymes via a TRAP-, AntiTRAP-dependent mechanism [Babitzke and Gollnick, 2001; Valbuzzi and Yanofsky, 2001; Yang and Yanofsky, 2005]. In this study, we have analyzed the global gene expression profile of B. subtilis in response to ammonium and tryptophan starvation using DNA microarray hybridization and 2D gel electrophoresis. The results allowed the 122

J Mol Microbiol Biotechnol 2007;12:121–130

classification of starvation induced genes into ammonium or tryptophan starvation specifically induced genes, genes that are induced by both and generally starvation induced genes. Many induced genes encoding proteins of so far unknown functions could be classified and seem to be involved in coping with ammonium and/or tryptophan starvation.

Results and Discussion

1. Transcriptome and Proteome Analyses in Response to Ammonium and Tryptophan Starvation In the ammonium- or tryptophan-limited minimal medium, cells stopped growth at an OD500 of 1.0. For transcriptome analyses, the RNA was checked for quality by Northern blots with ureC- or oppA-specific RNA probes for the ammonium starvation experiments and a trp-specific RNA probe for the tryptophan starvation experiments. As expected, these operons showed a strong transcriptional induction during the transition from exponential growth to stationary phase under the respective conditions (data not shown). The RNA samples taken during the growth (OD500 = 0.4) and at the transition phase were used for the microarray experiments. The protein samples labeled during the exponential growth, at transition and at 10, 30 and 60 min after the transition phase, were used for the proteome analyses. The transcriptome analyses revealed the significant induction (63-fold) of 110 genes and the repression (^0.3-fold) of 47 genes after ammonium starvation. In response to tryptophan starvation, 136 genes were upregulated and 172 genes were downregulated during the transition phase (for tables 1 and 2, see http://microbio1. biologie.uni-greifswald.de/publications.html). The accompanying proteome analyses revealed the induction of 76 proteins in ammonium-starved cells and 47 induced proteins in response to tryptophan starvation (fig. 1A, B). The expression data for upregulated genes and/or proteins are summarized in table 1 and the results for the specific, overlapping and general responses to ammonium and tryptophan starvation are discussed below. 2. Classification of Genes Induced Specifically in Response to Ammonium Starvation 2.1. Specific Induction of the TnrA, GlnR and L (BkdR/RocR) Regulons TnrA, GlnR: TnrA activates the transcription of at least 25 operons under nitrogen-restricted conditions [Fisher and Débarbouillé, 2002; Yoshida et al., 2003]. In Tam/Eymann/Antelmann/Albrecht/ Hecker

A

B

pI 7

4

pI 7

4

Fig. 1. Dual-channel images of the protein synthesis patterns of B. subtilis during exponential growth (green images) and under ammonium (A) or tryptophan (B) starvation (red images). The protein synthesis pattern (autoradiograms) in response to different times of starvation (t0 and 10, 30, 60 min later [1, 2, 3, 4]) were combined to generate fused proteome maps for ammonium or tryptophan starvation using the union image fusion approach of the Delta2D software. Proteins that are synthesized at increased levels (induction 63-fold) in response to ammonium or tryptophan starvation in at least two independent experiments are indicated by white labels. Their respective induction ratios for the different time point (1, 2, 3, 4) are listed in table 1. Spot identification was performed using MALDI-TOF-TOF mass spectrometry from Coomassie-stained 2D gels as described. Note, the proteins KamA, AsnO and SpoVT are induced in Coomassie-stained 2D gels but could be not detected by L-[35S]methionine labeling.

our analysis, the expression of twelve TnrA-dependent transcriptional units was found to be induced specifically by ammonium starvation since enhanced transcription of asnZ, comGC, comGG, gabP, nasA, nasBCDEF, nrgAB, tnrA, ykzB-ykoL, yrbD, ywrD and increased protein synthesis of AsnZ, PucL and TasA was only observed in ammonium- but not in tryptophan-starved cells. The nrgAB operon encoding an ammonium transporter and a nitrogen-regulated PII-like protein was most strongly induced [Wray et al., 1994; Detsch and Stülke, 2003]. In contrast, the TnrA-dependent genes ureABC, oppA and cah showed significant inductions at the transcriptional and/or translational levels after both, ammonium and tryptophan starvation which indicate a more complex

regulation (supplemental table 1, TnrA-regulon, fig. 1A, B, see also 4.1). In case of the ure operon, the transcriptional induction was significantly higher after ammonium starvation (ureA: 25- to 30-fold) compared to tryptophan starvation (ureA: 4-fold) and hence more specific for ammonium starvation. This difference in expression was also reflected on the proteome level (table 1). The transcription of the ure operon is controlled by three different promoters which are regulated by additional factors. The significantly higher induction by ammonium starvation might be caused by the TnrA activation and GlnR derepression of the ure P3 promoter [Wray et al., 1997; Brandenburg et al., 2002]. To verify this, Northern blot hybridization

B. subtilis Ammonium and Tryptophan Starvation Response


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Fig. 2. Transcript analysis of the ureABC

operon in response to ammonium (N) and tryptophan starvation (T). For Northern blot experiments, 5 g RNA each were applied from exponentially growing cells (Nc and Tc) and ammonium- or tryptophan-starved cells (N1–N5 and T1–T4). N1, 2, 3, 4, 5 are related to the transient phase and 10, 20, 30 and 60 min after the transition to stationary phase caused by ammonium starvation. T1, 2, 3, 4 are related to 20 and 10 min before transient point, transient point and 10 min after the transition to stationary phase caused by tryptophan starvation. The ladder of the RNA standard and the sizes of the different ure transcripts are indicated. In addition, the transcriptional organization of the ure operon is shown.

kb Nc N1 N2 N3 N4 N5

T1

T2

T3

T4

6. 95 4. 74

_ ~5 kb (ureABC-ywnA-B?)

2. 66

_ 3.2 kb (P3 ureABC) _ 2.4 kb (P2 ureABC)/23 S RNA _ 16 S RNA

1. 82 1. 52 1. 05 P3: A/TnrA/GlnR/CodY P2: H/CodY/? ur e A ur e B

with a ureA-RNA probe was performed showing a significantly induced transcript of 3.2 kb length after ammonium starvation only that probably initiates at the P3 promoter. Surprisingly, the bulk of the induced ureABC mRNA after ammonium starvation resulted from the H-dependent P2 promoter that is controlled by CodY [Wray et al., 1997]. This promoter is exclusively used for transcription induced by tryptophan starvation (fig. 2). The negatively TnrA-regulated glnRA operon was specifically induced, probably by derepression from GlnR, after ammonium starvation as demonstrated by transcriptome and proteome analyses (fig. 1A, table 1) which is consistent with previous studies [Fisher and Débarbouillé, 2002; Jürgen et al., 2005]. Besides the glnRA operon, a putative aspartate aminotransferase (yurG) was specifically induced by ammonium starvation which is also under negative TnrA control [Yoshida et al., 2003]. It is unknown whether GlnR derepression is responsible for yurG induction. L (BkdR/RocR): The bkd operon involved in degradation of branched chain amino acids and the rocABC and rocDEF operons, required for the catabolism of arginine and ornithine, were induced specifically by ammonium starvation. The 54-type sigma factor L and specific activators (BkdR, RocR) are required for the substrate-dependent induction of the bkd and roc operons in response to nitrogen starvation [Débarbouillé et al., 1991, 1999; Gardan et al., 1995, 1997] (table 1, L [BkdR/RocR]). In contrast, the glutamate dehydrogenase encoding rocG gene that catalyses the final step in arginine degradation was not induced. It has been shown that a high activity of 124

Tc


ur e C

y wnA

y wnB

2. 4 (2. 7) kb 3. 2 kb ~5 kb

glutamate dehydrogenase is incompatible with activity of TnrA [Belitsky and Sonenshein, 2004]. While the degradation of branched chain amino acids was induced, its biosynthesis was repressed upon ammonium starvation as indicated by the significant repression of the ilv-leu operon (supplemental table 2). This repression seems to be mediated by an active TnrA protein and is therefore specific for ammonium starvation [Tojo et al., 2004]. 2.2. Specific Induction of Carbon Catabolite-Controlled Genes The expression of the roc operons is also under CcpAdependent glucose repression [Yoshida et al., 2001]. Further carbon catabolite-repressed genes that were induced specifically by ammonium starvation, include the CcpAdependent genes acsA (acetyl-CoA synthetase), citB (aconitase) and dhaS (aldehyde dehydrogenase) as well as the CcpA-independently glucose-repressed genes glmS and iolS (table 1). Interestingly, the TCA cycle enzyme CitB was specifically induced by ammonium starvation. It was shown recently that citB expression is activated in the absence of glutamine or one of both equivalent substances, glutamate or ammonium. This activation seems to depend at least partly on TnrA [Blencke et al., 2006]. The induction of carbon catabolite-repressed genes by ammonium starvation might indicate a link between carbon and nitrogen metabolism. 2.3. Specific Induction of the alsSD Operon For the first time we could show that the alsSD operon as well as alsR encoding the operon-specific regulator Tam/Eymann/Antelmann/Albrecht/ Hecker

A

B. subtilis 168 were grown under ammonium and tryptophan starvation and the number of spores in comparison to the viable counts were determined at different time points as described in the Methods section.

2

2

1

1 1 0.1

0.01

1 0.1

0 1 2 3 4 5 6 7 8 9 10 11 Time (h)

were induced specifically upon ammonium starvation. The -acetolactate synthase AlsS condenses two molecules of pyruvate to -acetolactate that is converted to acetoin by the acetolactate decarboxylase AlsD. In B. subtilis, the acetohydroxy acid synthase (IlvBN) as well as AlsS are involved in the biosynthesis of acetolactate, a central metabolite, involved in both anabolism and catabolism. The induction of the alsSD operon in ammonium-starved cells might direct the acetolactate flux towards catabolism [Goupil-Feuillerat et al., 1997; Monnet et al., 2003]. Together with the bkd and ilv-leu operons, the degradation of acetolactate to acetoin by the alsSD operon might control the concentration of branched chain amino acids in nitrogen-starved cells. The mechanism for the specific transcriptional activation upon ammonium starvation is unknown as well as the fate of acetoin. Ye et al. [2000] observed an induction by anaerobiosis that was further increased by the addition of pyruvate. It might be possible that the pyruvate pool is increased in ammonium-starved cells caused by the degradation of certain amino acids (e.g. branched chain amino acids, cysteine and alanine). 2.4. Specific Induction of Sporulation Genes (F-, E-Regulons) The proteome analysis revealed the strong induction of early sporulation proteins belonging to the presporespecific F regulon (e.g. SpoIIQ [18-fold], SpoVT) and the mother cells-specific E regulon (e.g. AsnO, PrkA [71fold], SafA [53-fold], SpoIVA [128-fold], YaaH [105-fold]) at later time points (t3 and t4) in ammonium-starved cells [Errington, 2003; Kroos and Yu, 2000]. In addition, the sporulation frequency was increased solely in ammonium-starved cells after 6 h of stationary phase (fig. 3) inB. subtilis Ammonium and Tryptophan Starvation Response

B

10

0.01

0

Sporulation frequency (%)

Fig. 3. Determination of sporulation frequencies during ammonium (A) and tryptophan (B) starvation conditions. Cells of

OD500

10

1 2 3 4 5 6 7 8 9 10 11 Time (h)

Growth Sporulation frequency (%)

dicating that ammonium starvation triggers the sporulation process to a very manifested degree. The induction of the E-dependent putative aminotransferase (patB) during the transition phase upon ammonium starvation might be caused by another regulator because E is not yet active. PatB could be involved in the degradation of cysteine under nitrogen-restricted conditions, since cystathione -lyase and cysteine desulfhydrase activities were measured in vitro [Auger et al., 2005]. 2.5. Specific Induction of Other Genes in Response to Ammonium Starvation The alaR-T-yugI operon encoding a putative alanine transaminase (alaT) and the cypX gene encoding a cytochrome P450-like enzyme were induced specifically by ammonium starvation. In addition, 18 genes of unknown functions were induced specifically by ammonium starvation which could perform specific functions to cope with ammonium starvation. For example, YjbC is similar to N-acetyltransferases, YcbU is similar to aminotransferases, YjbG might function as oligopeptidase, and YurU, YuxJ and YwoE share similarities to transporters (table 1). 3. Specific Responses to Tryptophan Starvation 3.1. Specific Induction of Tryptophan Biosynthesis Enzymes (TRAP Regulon) The transcriptome and proteome analyses revealed the specific induction of about 60 genes in response to tryptophan starvation. These include the trp operon (trpEDCFBA), a suboperon within the aromatic supraoperon and the trpG gene (pabA), located in the unlinked folate operon. These 7 genes encode enzymes required for J Mol Microbiol Biotechnol 2007;12:121–130

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the biosynthesis of tryptophan from chorismic acid, the common aromatic amino acid precursor. The transcription of these genes is regulated by the trp RNA-binding attenuation protein (TRAP) in response to the accumulation of tryptophan [Babitzke and Gollnick, 2001; Gollnick et al., 2002]. Furthermore, the TRAP-regulated yczA-ycbK-(at) operon encoding the anti-TRAP protein (AT) and the TRAP-regulated tryptophan transporter encoded by yhaG were induced specifically by tryptophan starvation [Sarsero et al., 2000a, b]. In addition to the TRAP-regulated genes, the hisC-tyrA-aroE upstream region of the supraoperon involved in chorismic acid biosynthesis as well as the tryptophanyl-tRNA synthetase gene trpS represent tryptophan starvation specifically induced genes. It has been shown that the transcription of trpS is also regulated by the same T-box antitermination mechanism like the at operon [Sarsero et al., 2000a; Valbuzzi and Yanofsky, 2001]. 3.2. Specific Induction of Other Genes in Response to Tryptophan Starvation The RelA-dependent genes adeC, ald, hpr and yetH, the degSU two-component system for degradative enzyme production, the NADH dehydrogenase encoded by ndhF and the negative sporulation regulatory phosphatase spo0E were among the tryptophan starvation specifically induced genes (table 1, fig. 1B). In addition, the transcription of 25 y-genes encoding proteins of unknown function was enhanced after tryptophan starvation only including yodF and yusM that encode a putative proline permease and dehydrogenase, respectively. The proteome analysis revealed elevated protein synthesis of the putative ABC transporter ATP-binding protein YurY as well as YybI (unknown) after tryptophan starvation only. It might be that these y-genes code for proteins that are necessary for the specific adaptation to tryptophan starvation. 4. Genes Induced by both Ammonium and Tryptophan Starvation 4.1. Overlapping Responses That Are Different from CodY, RelA and H Ammonium starvation leads to amino acid depletion (e.g. glutamine), causing responses that are also typical for amino acid starvation. Furthermore, both conditions result in the transition to stationary phase. This might cause the common induction of genes by both ammonium and tryptophan starvation. These genes could be classified into (1) overlapping genes involved in rather specific functions and (2) more generally starvation in126


duced genes that overlap also with other starvation conditions such as glucose or phosphate starvation. The second group includes the CodY and H general starvation regulons as well as the RelA-dependent stringent response (see 4.2). The first group consists of 25 genes that were induced at similar levels in response to ammonium and tryptophan starvation. These genes include the cephalosporin C deacetylase (cah, TnrA-group), the CcpA-repressed citrate synthase CitZ [Kim et al., 2002], the putative D-alanine aminotransferase dat, the maeN gene encoding a Na+/malate symporter and the two-component system YufLM involved in regulation of maeN [Tanaka et al., 2003]. In addition, 12 further y-genes including the putative 1-pyrroline-5-carboxylate dehydrogenase ycgN and the ribosomal-protein-alanine N-acetyltransferase yjcK were induced after ammonium and tryptophan starvation (table 1). The corresponding gene products could be required for the adaptation of the cell to ammonium and tryptophan starvation, e.g. by the generation of glutamate (ycgN, dat), modification of ribosomal proteins (yjcK) and uptake of malate (maeN). 4.2. General Induction of the CodY, RelA and H-Regulons by Ammonium and Tryptophan Starvation The transcriptome and proteome analyses revealed the general induction of 18 CodY-regulated transcriptional units in response to ammonium and tryptophan starvation, as well (table 1, CodY regulon). This indicates a CodY derepression in response to both starvation conditions. The decrease of GTP caused by the stringent response might be responsible for the CodY derepression under ammonium and tryptophan starvation conditions [Freese et al., 1979; Ratnayake-Lecamwasam et al., 2001; Inaoka and Ochi, 2002; Inaoka et al., 2003]. In ammonium-starved cells, the decrease of branched chain amino acids, caused by the induction of the bkd operon and the repression of ilv-leu operon, might also be involved in CodY derepression. It was shown previously that CodY binds GTP and/or branched chain amino acids [Blagova et al., 2003; Shivers and Sonenshein, 2004]. Interestingly, the CodY-dependent ABC transporter YurJ was 6-fold higher induced in tryptophan-starved cells than after ammonium starvation (table 1) which might be caused by the absence of active TnrA in tryptophan-limited cells. A negative influence of TnrA on the transcription of yurJ was found in nitrogen-restricted cells [Yoshida et al., 2003].

Tam/Eymann/Antelmann/Albrecht/ Hecker

The induction of 11 positively RelA-controlled genes (e.g. spo0A, ytzE, yvyD) (online supplement table 1, www.karger.com/doi/10.1159/000096467, RelA regulon) and the repression of at least 70 negatively RelA-dependent genes (e.g. frr, rplA,C,D,W, rpsJ, tsf ) in response to ammonium and tryptophan starvation are indicative for the stringent response (online supplement table 2, www.karger.com/doi/10.1159/000096467). In addition to the stringently controlled genes which are partly co-regulated by H [Drzewiecki et al., 1998; Eymann et al., 2001], 11 H-dependent transcriptional units (e.g. phrG-, spoIIA operon, ymaH ) were induced after both starvation conditions (table 1, H regulon). The corresponding gene products of CodY-, RelA- and H-regulated genes could perform functions necessary for coping with both, ammonium and tryptophan starvation, e.g. hydrolysis of amino acids (yhdG, amhX), transport of oligo- and dipeptides (app, dpp) and unknown substances (yufOPQ, yurJ), asparagine synthesis (asnH) and synthesis of Rap phosphatases (rapA,C,G) as well as their specific regulators (phrA,C,G) [Perego, 1997; Jiang et al., 2000]. Rap phosphatases can dephosphorylate Spo0FP, a member of the phosphorelay signal transduction system that governs the initiation of sporulation [Hoch, 1993]. Thus, they provide access for negative signals to influence the cell’s decision of whether to initiate the sporulation or not [Lazazzera et al., 1999].

Conclusions

The responses of B. subtilis to ammonium or tryptophan starvation involve major changes in the global gene expression pattern. Both starvation conditions caused the common induction of the CodY, RelA and H general starvation regulons and overlapping responses that are independent from CodY, RelA, H. The CodY and H general starvation regulons are required for the adaptation of the cell to nutrient depletion and to post-exponential stationary phase processes such as survival under non-growing conditions, competence or sporulation. The negative RelA-dependent stringent response consisting of the repression of components of the translational apparatus including ribosomal proteins and translation factors is characteristic for the non-growing state under all starvation conditions. In contrast, clearly ammonium- or tryptophan starvation-specific gene expression patterns were detected.


The specific response to ammonium starvation includes mainly the induction of TnrA- and L(BkdR/ RocR)-dependent genes that are involved in the highaffinity uptake of ammonium and utilization of alternate nitrogen sources such as amino acids (e.g. asparagine, branched chain amino acids, arginine, alanine), -aminobutyrate, nitrate/nitrite, uric acid/urea and oligopeptides and the induction of glutamine synthetase (GlnA). Furthermore, the transcriptome analysis provided evidence for the synthesis of glutamate as the second substrate for GlnA in addition to ammonium. This is demonstrated by the upregulation of glutamate generating enzymes such as aspartate aminotranferase (yurG), ornithine aminotransferase (rocD), alanine transaminase (alaT), other potential -ketoglutarate utilizing aminotransferases (ycbU, patB), -glutamyltransferase (ywrD) as well as aconitase (citB) that is involved in the synthesis of -ketoglutarate/glutamate. Alternate nitrogen sources such as -aminobutyrate (gabP) or their degradation products, e.g. aspartate (asnZ) might function as amino group donors for aminotransferases. Glutamate is also generated by degradation of arginine/ornithine as indicated by the specific induction of the rocA,D,F genes. These reactions and the prevention of rocG expression indicate that the level of glutamate as substrate for glutamine synthetase and nitrogen donor for transaminations is critical and should be maintained. The asparagine synthetase AsnO was synthesized specifically in ammonium-starved cells. In addition to GlnA, the asparagine synthetase AsnO might be involved in ammonium assimilation during ammonium starvation. In contrast, the CodYdependent asnH gene encoding another asparagine synthetase was induced in response to both, ammonium and tryptophan starvation as well [Molle et al., 2003; Yoshida et al., 1999]. The specific response of B. subtilis to tryptophan starvation includes the TRAP regulon involved in tryptophan biosynthesis (e.g. trp operon) as well as genes involved in the generation of ammonia (adeC, ald) and glutamate (yodF, yusM) that are different from that induced by ammonium starvation. Moreover, the ald and yodF genes are negatively controlled by TnrA preventing an induction during ammonium starvation [Yoshida et al., 2003]. Induction of adeC and ald is rather specific for amino acid starvation since these are also induced by norvaline in a RelA-dependent manner [Eymann et al., 2002]. The gene products of the yodF and yusM genes as well as the gene product of ycgN could be involved in the uptake and utilization of proline thereby generating gluJ Mol Microbiol Biotechnol 2007;12:121–130

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tamate as nitrogen donor for transaminations in amino acid biosynthesis. In summary, this paper present one further step towards the description of the responses of B. subtilis to ammonium and tryptophan starvation by the use of transcriptome and proteome analyses. Several genes with still unknown function were induced by ammonium and/or tryptophan starvation which might be required for the adaptation of the cell to these starvation conditions. Thus, it is subject to future studies to characterize the functions of these unknown genes during ammonium and/or tryptophan starvation conditions.

Experimental Procedures Bacterial Strains and Culture Conditions B. subtilis wild-type 168 (trpC2) was grown aerobically at 37 ° C under vigorous agitation in Belitsky minimal medium (BMM) as described previously [Stülke et al., 1993]. For ammonium or tryptophan starvation, cells were grown in BMM containing 0.7 mM instead of 15 mM (NH4)2SO4 or 4 M instead of 80 M tryptophan, respectively. In each starvation experiment the stationary phase was reached at an optical density at 500 nm (OD500) of about 1. Assay for Sporulation Frequency Cells grown under ammonium and tryptophan starvation conditions were diluted at different times along the growth curve in 0.9% NaCl solution and aliquots of appropriate dilutions were incubated at 80 ° C for 10 min to kill the cells. All appropriate dilutions containing either viable cells or survived spores were plated on LB plates, incubated overnight at 37 ° C and counted for colony-forming units that indicates the number of spores. The number of spores was related to the number of viable cells at each time point. Preparation of the Cytoplasmic L-[35S]Methionine-Labeled Protein Fraction Cells grown in minimal medium were pulse-labeled for 5 min each with 10 Ci of L-[35S]methionine per ml at an OD500 of 0.4 (for control) and at several time points (transition point and 10, 30 and 60 min) after the transition into the stationary phase caused by ammonium or tryptophan starvation. The soluble protein fractions were prepared as described previously [Bernhardt et al., 1999]. Two-Dimensional (2D) Gel Electrophoresis Determination of protein content, separation of 80 g of the L-[35S]methionine-labeled protein extracts by two-dimensional gel electrophoresis (2D-PAGE), silver nitrate staining, exposition to Phosphor screens (Molecular Dynamics, Sunnyvale, Calif., USA) and out reading with a Phosphor Imager SI instrument (Molecular Dynamics) were done as described previously [Bernhardt et al., 1999]. For identification of the proteins by mass spectrometry, non-radioactive protein samples of 200 g were separated by preparative 2D-PAGE and stained with Colloidal Coomassie Brilliant Blue (Amersham Biosciences) as described by Eymann et al. [2004].

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Quantitative Image Analysis Quantitative image analysis was performed with the Decodon Delta2D software, version 3.3 (http://www.decodon.com) which is based on the dual-channel image analysis technique [Bernhardt et al., 1999]. Creation of fused 2D gels of the ammonium or tryptophan starvation experiments, spot detection and normalization were performed as described by Tam et al. [2006]. Proteins showing an induction of at least 3-fold compared to the control during the L-[35S]methionine pulse in two independent experiments were designated as significantly induced proteins after ammonium or tryptophan starvation, respectively. Protein Identification by MALDI-TOF-TOF Mass Spectrometry Spot cutting from Colloidal Coomassie-stained 2D gels, tryptic digestion of the proteins, spotting of the resulting peptides onto MALDI targets, MALDI-TOF-TOF measurement of spotted peptide solutions and protein identification was performed as described previously [Eymann et al., 2004]. Northern Blot Experiments Total RNA of B. subtilis wild-type was isolated from cells during the exponential growth (for control) and after the transition into the stationary phase provoked by ammonium or tryptophan starvation as described [Jürgen et al., 2005]. Northern blot analyses were performed as described previously [Wetzstein et al., 1992]. Hybridizations specific for ureA/C, oppA and trpB were conducted with the digoxigenin-labeled RNA probes synthesized in vitro with T7 RNA polymerase from T7 promoter containing internal PCR products of ureA/C, oppA or trpB. The following primers were used for PCR, respectively: ureA-for (5-ATGAAACTGACACCAGTTGAAC-3) and ureA-rev (5-CTAATACGACTCACTATAGGGAGA/TGACTTCACCTCCGCAGAAA-3); ureC-for (5-ACGGATTTATGGATCGAAGTC3) and ureC-rev (5-CTAATACGACTCACTATAGGGAGAGATGATGTCATGCTGATCGC-3); oppA-for (5-AATGATTCAGTATCAGGCGG-3) and oppA-rev (5-CTAATACGACTCACTATAGGGAGATACTGCTTGAGCGATTTTCG-3); trpB-for (5-GGAAACACTCATGCAGCCG-3) and trpB-rev (5-CTAATACGACTCACTATAGGGAGATCCACCGCTTCTTCATCGG-3). Transcriptome Analysis by DNA Microarray Hybridization Determination of RNA concentration and quality, generation of fluorescence-labeled cDNA and hybridization with B. subtilis whole-genome microarrays (Eurogentec), quantification of hybridization signals, background subtraction was performed as described previously [Jürgen et al., 2005]. Calculation of normalized intensity values and ratios for the two dyes were performed with the Genespring software from Agilent Technologies (version 7.1). Induced and repressed genes with an expression level ratio of 63 or ^0.33 in both independent experiments respectively were regarded as significant genes. Final evaluation of the microarray data included the consideration of putative operons derived from the genome sequence, using the SubtiList database (http://genolist.pasteur.fr/SubtiList/) as well as previously known transcriptional units. The induced genes/proteins were classified according to previously described regulons including TnrA [Fisher and Débarbouillé, 2002; Yoshida et al., 2003], L [Débarbouillé et al., 1999; Gardan et al., 1995], TRAP


[Babitzke and Gollnick, 2001; Sarsero et al., 2000a, b], RelA [Eymann et al., 2002], H [Lee and Price, 1993; Stover and Driks, 1999; McQuade et al., 2001; Britton et al., 2002], CodY [Lazazzera et al., 1999; Molle et al., 2003], F [Steil et al., 2005], E [Feucht et al., 2003; Eichenberger et al., 2003, Steil et al., 2005], B [Petersohn et al., 2001], and carbon catabolite control [Renna et al., 1993; Yoshida et al., 2001; Moreno et al., 2001; Blencke et al., 2003] using the Genespring software from Agilent Technologies (version 7.1).

Acknowledgements We thank Britta Jürgen and Stefanie Leja for the help with the microarray experiments, Ulrike Mäder for help with data analysis and critical reading of the manuscript, and Decodon GmbH for support with the Decodon Delta2D software. This work was supported by a scholarship of the ‘Ministry of Education and Training of Viet Nam’ (MOET) to L.T.T., and grants from the ‘Deutsche Forschungsgemeinschaft’ (DFG), the ‘Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie’ (BMFT), the ‘Fonds der Chemischen Industrie’ and Genencor International, Inc. (Palo Alto, Calif., USA) to M.H.

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