Genomewide transcriptional changes associated with genetic alterations and nutritional supplementation affecting tryptophan metabolism in Bacillus subtilis Randy M. Berka*, Xianju Cui*, and Charles Yanofsky†‡ *Novozymes Biotech, Inc., Davis, CA 95616; and †Department of Biological Sciences, Stanford University, Stanford, CA 94305 Contributed by Charles Yanofsky, March 19, 2003 DNA microarrays comprising Ϸ95% of the Bacillus subtilis anno- initiation at the trp operon promoter͞operator. Transcription tated protein coding ORFs were deployed to generate a series of initiation is not known to be regulated at the trp operon snapshots of genomewide transcriptional changes that occur when promoter of B. subtilis. Transcription of the structural genes in cells are grown under various conditions that are expected to the trp operons of both organisms is regulated by transcription increase or decrease transcription of the trp operon segment of the attenuation, but by different mechanisms. In B. subtilis a tryp- aromatic supraoperon. Comparisons of global expression patterns tophan-activated RNA-binding protein (TRAP), the product of were made between cells grown in the presence of indole acrylic the mtrB gene, regulates attenuation. The mtrB coding sequence acid, a specific inhibitor of tRNATrp charging; cells deficient in resides in a two-gene operon with mtrA, which specifies GTP expression of the mtrB gene, which encodes the tryptophan- cyclohydrolase I, the enzyme catalyzing the first step in pterin activated negative regulatory protein, TRAP; WT cells grown in the formation in folic acid biosynthesis. In addition to these aro- presence or absence of two or three of the aromatic amino acids; matic-folate cross-pathway features, at least one additional and cells harboring a tryptophanyl tRNA synthetase mutation operon, rtpA-ycbK, appears to play an important regulatory role conferring temperature-sensitive tryptophan-dependent growth. in trp operon expression in B. subtilis (4, 5). Transcription of Our findings validate expected responses of the tryptophan bio- rtpA-ycbK is regulated by the T box antitermination mechanism synthetic genes and presumed regulatory interrelationships be- in response to a deficiency of charged tRNATrp (4, 6). Expression tween genes in the different aromatic amino acid pathways and of the rtpA-ycbK operon leads to the synthesis of the anti-TRAP the histidine biosynthetic pathway. Using a combination of super- regulatory protein AT, the rtpA gene product (5). AT can vised and unsupervised statistical methods we identified Ϸ100 inactivate TRAP and when it does this allows trp operon genes whose expression profiles were closely correlated with transcription and trpG translation. AT production is also regu- those of the genes in the trp operon. This finding suggests that lated translationally, in response to the accumulation of un- expression of these genes is influenced directly or indirectly by charged tRNATrp (G. Chen and C.Y., unpublished data). Many regulatory events that affect or are a consequence of altered of the known B. subtilis genes that are presumed to play a role tryptophan metabolism. in aromatic amino acid metabolism and folic acid synthesis and regulation are presented in Fig. 1. omologous protein domains are used by Bacillus subtilis and B. subtilis also lacks a structural homolog of TyrR, the major HEscherichia coli to catalyze the same reactions in the regulatory protein of E. coli that controls expression of the biosynthesis of the aromatic amino acids (1). Despite this common aromatic pathway genes and the genes required for similarity, very different regulatory proteins and mechanisms are phenylalanine and tyrosine synthesis. However, some of these B. used by these bacteria to regulate aromatic amino acid synthesis. subtilis genes are regulated in response to tyrosine or phenylal- These differences must be partly caused by the different evolu- anine accumulation. In addition, in B. subtilis, a single gene, tionary histories and experiences of these microorganisms. aroA, specifies 3-deoxy-D-arabino-heptulosonate-7-phosphate Operon organization also is somewhat different in the two (DAHP) synthase, the enzyme that catalyzes the first step in the species, reflecting regulatory interrelationships described as common aromatic pathway. Synthesis of this enzyme is regulated cross-pathway control, that exist between genes for different only indirectly by aromatic amino acids (1, 3). E. coli produces pathways in B. subtilis that are not evident in E. coli. Thus, the three nearly identical DAHP synthases that catalyze this reac- six-gene trp operon of B. subtilis resides within an aromatic tion, and each is subject to transcriptional regulation principally supraoperon that contains six additional genes, three upstream by a different aromatic amino acid. and three downstream, concerned with the common aromatic The present study parallels a similar investigation of E. coli pathway and with phenylalanine, tyrosine, and histidine biosyn- genes that respond transcriptionally to culture conditions and thesis (Fig. 1). The seventh trp gene, trpG (pabA), is in the folate genetic alterations that influence tryptophan metabolism (7). operon. This gene specifies a protein that functions both in Herein we describe the application of B. subtilis DNA microar- tryptophan and folate biosynthesis; presumably because of this, rays as an initial step toward our goal of determining the global it is subject to regulation by both metabolites. In E. coli the effects on gene expression of varying tryptophan and charged five-gene trp operon encodes all seven protein domains needed tRNATrp availability in B. subtilis. for tryptophan biosynthesis; two of these genes encode fused protein domains that engender bifunctional polypeptides (2). Materials and Methods The B. subtilis aromatic supraoperon has three promoters as B. subtilis Strains. The following B. subtilis strains were used in shown in Fig. 1 (1, 3). One promoter is located before the three these studies: CYBS400 (WT) (4), CYBS222 (mtrBϪ) [a frame- genes upstream of the six-gene trp operon. A second promoter shift mutation located near the end of the mtrB coding region immediately precedes the trp operon segment. These two pro- yields a TRAP protein with reduced activity (3)], and BS1A353 moters provide trp operon transcripts. The third supraoperon promoter is within trpA, the last trp gene; it provides transcripts derived from the last three genes of the supraoperon. A major Abbreviations: TRAP, tryptophan-activated RNA-binding protein; PC, principal compo- regulatory difference between E. coli and B. subtilis is that E. coli nent; PCA, PC analysis. uses a DNA-binding repressor protein to control transcription ‡To whom correspondence should be addressed. E-mail: [email protected]. 5682–5687 ͉ PNAS ͉ May 13, 2003 ͉ vol. 100 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1031606100 Downloaded by guest on September 24, 2021 Table 1. B. subtilis cells and growth conditions used as sources of RNA for microarray studies Experiment B. subtilis strains and culture conditions Replicates* 1 WT in minimal medium versus WT in 8 minimal ϩ 50 ␮g͞ml tryptophan WT grown in minimal medium ؉ 30 ␮g͞ml 6 2 indole acrylic acid versus WT grown in minimal medium 3 mtrB-deficient mutant grown in minimal 8 medium ؉ 50 ␮g͞ml tryptophan versus WT grown in minimal medium ϩ 50 ␮g͞ml tryptophan 4 WT grown in minimal medium with 50 5 ␮g͞ml each phenylalanine and tyrosine versus WT grown in minimal medium 5 WT grown in minimal medium versus WT 7 grown in minimal medium with 50 ␮g͞ml Fig. 1. Known relationships among the genes of the aromatic supraoperon. each tryptophan, phenylalanine, and Colored lines connect the genes that are directly responsible for synthesis of tyrosine tryptophan (red), folate (blue), histidine (green), phenylalanine (gray), cho- 6 WT grown in minimal medium with 50 7 rismate (violet), and tyrosine (brown). Promoters are denoted by PЈ. Orange ␮g͞ml each phenylalanine and tyrosine rectangles represent leader regulatory regions controlled by tRNA-mediated versus WT grown in minimal medium antitermination, and gray boxes define regions at which TRAP (mtrB gene with 50 ␮g͞ml each tryptophan, product) binds and regulates translation. The promoter of the trp operon phenylalanine, and tyrosine itself (trpEDCFBA) is denoted as a violet box because TRAP regulates tran- ts scription at this site. Anti-TRAP (rtpA gene product) forms a complex with 7 trpS1 mutant grown at 38°C in minimal 8 ؉ TRAP and inhibits its activity and is noted as AT. medium 0.2% acid hydrolyzed casein ؉ 50 ␮g͞ml tryptophan versus WT grown in the same medium (trpS1), a mutation in the tryptophanyl-tRNA synthetase struc- To minimize intensity biases that are sometimes observed with the use of tural gene resulting in temperature-sensitive tryptophan- Cy3͞Cy5 dyes we used a dye-swapping strategy for some of the experiments dependent growth (8). listed. Consequently, the fluorescence intensity ratios were calculated as the intensity derived from one cDNA probe (growth condition or strain) divided by Culture Conditions and Isolation of RNA. Cultures (50 ml) were the other. The inducing condition, indicated in boldface type, corresponds to grown to midlog phase with shaking in minimal medium (9) plus the cells indicated by * in Table 2. The term “replicates” as used here refers to trace elements, plus and minus various supplements, at 37°C. the number of times the B. subtilis genome was queried with fluorescently