JOURNAL OF BACrERIOLOGY, Jan. 1994, p. 108-114 Vol. 176, No. 1 0021-9193/94/$04.00+0 Copyright X) 1994, American Society for Microbiology The Nitrogen-Regulated Bacillus subtilis nrgAB Operon Encodes a Membrane Protein and a Protein Highly Similar to the Escherichia coli glnB-Encoded PI, Protein LEWIS V. WRAY, JR., MARIETTE R. ATKINSON, AND SUSAN H. FISHER* Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118 Received 3 September 1993/Accepted 28 October 1993 In order for cells to obtain nitrogen for macromolecular synthesis, nitrogen-containing compounds must be transported into the cell and, if necessary, degraded to either NH4' or glutamate. The expression of enzymes required for the utilization of nitrogen-containing compounds is generally induced by their substrates. In addition, the expression of many degradative and transport systems is regulated in response to nitrogen availability in the growth medium (31). In enteric bacteria, nitrogen regulation is mediated both transcriptionally and posttranslationally. The nitrogen regulatory (Ntr) system controls the expression of many genes involved in nitrogen metabolism, including glnA, the structural gene for glutamine synthetase (GS). Transcriptional activation of Ntr-regulated genes requires the phosphorylated form of the NR, (NtrC) protein (31). NR,-phosphate binds to a specific enhancer sequence and stimulates transcription initiation at promoters transcribed by RNA polymerase containing the C(x4 sigma factor (29). The NRI, (NtrB) protein functions as a kinase/phosphatase that determines the level of NR, phosphorylation (31). The enzymatic activity of GS can be reduced posttranslationally by the covalent attachment of AMP to the GS protein (adenylylation). In Escherichia coli, the glnE-encoded adenylyltransferase enzyme (ATase) catalyzes both the attachment and removal of the AMP group from GS (31). The enzymatic activities of both NRI, and ATase are modulated by the glnB-encoded PI, protein (31). The activity of PII is regulated posttranslationally by a reversible uridylylation which is catalyzed by the glnD-encoded uridylyltransferase/ uridylyl-removing enzyme (UT/UR). The PI, protein is maintained in an unmodified state during growth in the presence of excess nitrogen. Unmodified PI, stimulates NRI, phosphatase activity, resulting in decreased transcriptional activation of Ntr-regulated genes. In addition, the unmodified PI, protein stimulates the adenylylation activity of ATase, causing the adenylylation and inactivation of GS. During nitrogen-limited growth, UT/UR converts PI, to PI,-UMP. This allows NRI, to convert NRI to its transcriptionally active form, NR,-phos- phate. Deadenylylation of adenylylated GS by ATase is also stimulated by PI1-UMP. Homologs of PI, have been identified in microorganisms as diverse as archaebacteria (39) and cyanobacteria (41), although their exact roles in cellular metabolism have not yet been established. Nitrogen assimilation in Bacillus subtilis differs significantly from that seen in enteric bacteria. The enzymatic activity of the B. subtilis GS protein is not known to be regulated by adenylylation or any other posttranslational modification (9, 13). In addition, there is no evidence for a global nitrogen regulatory system analogous to the enteric Ntr system (14). The B. subtilis GS structural gene, glnA, lies within the glnRA operon (33). Expression of the glnRA operon is negatively regulated by the GlnR repressor during growth with excess nitrogen in response to an as yet unidentified metabolic signal (14, 33). The glnRA operon is transcribed by the vegetative (er) form of RNA polymerase. The B. subtilis homolog (uL) of the enteric o7" sigma factor is required for expression of a sucrose-degradative enzyme and for utilization of arginine, ornithine, isoleucine, and valine as sole nitrogen sources but does not control GS synthesis (8). In B. subtilis, enzymes whose expression is known to be derepressed during nitrogen-limited growth include GS (14), aspartase (40), asparaginase (2, 16), urease (2), and -y-aminobutyrate permease (2, 44). During growth in the presence of excess nitrogen, the expression of GS, urease, asparaginase, and y-aminobutyrate permease is repressed in wild-type cells, but not in glnA mutants (2, 34). This argues that the wild-type OS protein is required for signalling nitrogen availability and that nitrogen regulation appears to be mediated by a common signal in B. subtilis. The B. subtilis GS regulatory protein, GlnR, is not known to regulate the expression of any nitrogenregulated enzyme other than GS (2, 14). To investigate further the mechanisms mediating nitrogen regulation in B. subtilis, the genetic organization of another operon transcribed from a nitrogen-regulated promoter was determined. Unexpectedly, this operon, first identified by a Tn917-lacZ insertion mutation (2), was found to encode a protein whose amino acid sequence is similar to that of the E. coli glnB-encoded P,, protein. * Corresponding author. Fax: 617-638-4286. Electronic mail address: firstname.lastname@example.org. 108 Downloaded from http://jb.asm.org/ on October 25, 2017 by guest Expression of j8-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M. R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nmg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrg4B. The nrg4 gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli gInB-encoded PI, protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis crA-dependent promoters. NITROGEN-REGULATED BACILLUS SUBTILIS nrgAB OPERON VOL. 176, 1994 TABLE 1. B. subtilis strains used in this study Genotypea Strain 168 SF10 SF12 SF300 SF305 SF320 SF321 SF330 SF335 trpC2 Wild type nrg-29::Tn917-lacZ nrg-29::Tn917-lacZ trpC2 nrgA::pNRG305 erm trpC2 nrg-29::Tn917-lacZ::pTV20 cat nrg-29::Tn9J7-lacZ::pTV21A2 cat AamyE::lacZ cat trpC2 AamyE::'F(nrgA-lacZ)335 cat IlacZ Tn917 Reference, source, or derivation This laboratory 2 2 168 x SF12 DNA 168 x pNRG305 SF12 x pTV20 SF12 x pTV21A2 168 x pSFL1 168 x pNRG335 pNRG301, pSFN20 pNRG303, pSFN21 (4) ------ /7 pNRG342 pNRG305 pNRG325 pNRG323 trpC2 pNRG328 SF335 x SF368 DNA AnrgAB368::spec trpC2 168 X pNRG364 SF364 AnrgB364::spec trpC2 168 X pNRG365 SF365 4D(nrgB-lacZ)365 cat trpC2 168 X pNRG368 SF368 AnrgAB368::spec trpC2 aGenotype symbols are those of Anagnostopoulos et al. (1), with the addition SF364 SF348 AamyE::4(nrgA-lacZ)335 cat AnrgB364::spec trpC2 AamyE::4F(nrgA-lacZ)335 cat Ispec SF368 of nrg to denote nitrogen-regulated gene. MATERUILS AND METHODS Bacterial strains and plasmids. E. coli strains NM522 (17) and KE93, a derivative of MM294 which contains the pcnB80 (22) mutant allele, were used as hosts for DNA cloning experiments. Plasmid pJL73 contains a spectinomycin resistance gene cloned into the SmaI site of pBluescriptII SK (Stratagene). E. coli plasmid pJDC9 was designed to allow the cloning of DNA fragments with strong promoter activity and contains a polylinker cloning region flanked by transcriptional terminators (6). pJDC9 confers erythromycin resistance to both E. coli and B. subtilis, although it does not replicate in B. subtilis. The B. subtilis strains used in this study are listed in Table 1. Cell growth and media. Methods used for bacterial cultivation have been described previously (3). Glucose was added at 0.5%, and glutamate, proline, and NH4Cl were added at 0.2% to the MOPS (morpholine propanesulfonic acid) minimal medium of Neidhardt et al. (27). The growth phenotype of the nrgAB deletion mutants was examined on BSS minimal plates (5) made with Noble agar (Difco Laboratories, Detroit, Mich.). All nitrogen sources were filter sterilized and added at 0.2% to the BSS minimal plates. Spore production was examined in liquid cultures grown overnight in Difco sporulation medium (38) or in Sterlini-Mandelstam resuspension medium as previously described (28). Spore formation was measured by titration of the survivors of heating for 10 min at 80 to 85°C (28). Enzyme assays. 1-Galactosidase activity was assayed as previously described (3) in extracts of cultures grown to mid-log growth phase (70 to 90 Klett units) in MOPS minimal medium. One unit of ,B-galactosidase activity produced one nanomole of o-nitrophenol per minute. GS activity was determined by the Mn24-dependent reverse transferase assay in permeabilized cells (13). DNA cloning and plasmid constructions. Chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned by using the method described by Youngman (46). Plasmid pSFN20, which contains nrgAB DNA from the nrg-29:: Tn917-lacZ insertion site to the downstream EcoRI site (Fig. 1), was obtained by using the integrative plasmid pTV20 (46). Plasmid pNRG301 was constructed by subcloning an EcoRIBglII DNA fragment containing the downstream end of Tn917 r' SF365 nrg lacZ cat S FIG. 1. Physical structure of the nrgAB operon. The position of the nrg-29::Tn917-lacZ insertion is indicated at the top. The nrgAB promoter is indicated by the arrow to the left of the nrgA gene. A putative transcription terminator is indicated by the stem-and-loop structure to the right of the nrgB gene. The EcoRI site at the left is located approximately 15 kb upstream of the nrgAB operon. Physical maps of the cloned DNA inserts are shown below the map of the nrgAB operon. A deletion (A) in pNRG303 and pSFN21 is indicated. The chromosomal structures of strains SF364, SF365, and SF368 are shown at the bottom. The Tn917-lacZ transposon and lacZ cat gene cassette are not drawn to scale. and the adjacent nrgAB DNA from pSFN20 into pMTL20P (4). With the integrative plasmid pTV21A2 (46), pSFN21 was obtained by digesting chromosomal DNA from SF321 with EcoRI (Fig. 1). Plasmid pSFN21 contains 1.0 kb of DNA upstream of the nrg-29::Tn917-lacZ insertion. Southern blot analysis of B. subtilis chromosomal DNA revealed that the EcoRI site is located approximately 15 kb upstream of the nrg-29::Tn917-lacZ insertion (46). Since pSFN21 contains only 1 kb of B. subtilis chromosomal DNA, a deletion must have occurred in the B. subtilis chromosomal sequences present in the original clone. A plasmid containing the entire 15 kb of upstream DNA, pNRG342, was obtained by using the E. coli strain KE93 (pcnB80) as the cloning host. The pcnB80 mutation lowers the copy number of ColEl-derived plasmids 16fold (22) and thus facilitates the cloning of B. subtilis chromosomal DNA which is toxic in E. coli (46). Plasmid pNRG303 (Fig. 1) was constructed by subcloning a 1.3-kb EcoRI-BamHI DNA fragment containing the upstream end of Tn917 and the adjacent nrgA DNA from pSFN21 into pJDC9 (6). The nrgAB promoter was subcloned as a 300-bp TaqI fragment from pNRG303 into the AccI site of mpl8 (45). Plasmid pNRG305 (Fig. 1) was constructed by subcloning the EcoRI-HindIIl DNA fragment containing the nrgAB promoter from the M13 clone into pJDC9. B. subtilis SF305, which contains the plasmid pNRG305 integrated at the nrgAB chromosomal locus, was constructed by transforming strain 168 to Downloaded from http://jb.asm.org/ on October 25, 2017 by guest SF335 x SF364 DNA SF344 109 110 WRAY ET AL. RESULTS AND DISCUSSION DNA sequencing of the nrgAB operon. Chroand Cloning mosomal DNA adjacent to the B. subtilis nrg-29::Tn917-lacZ transposon insertion (2) was cloned in E. coli plasmids (see Materials and Methods). Additional clones were obtained by using an integrational plasmid, pNRG305, for chromosomal walking. Figure 1 shows the physical map of the DNA inserts for these clones. The nucleotide sequence obtained from these clones is presented in Fig. 2. Analysis of the DNA sequence revealed that the nrg-29::Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon that we have designated nrgAB. Both of the open reading frames are preceded by nucleotide sequences that are complementary to the 3' end of the B. subtilis 16S rRNA (25) and most likely serve as in vivo ribosome-binding sites. Immediately downstream of the second open reading frame (at nucleotides 1712 to 1734) is an inverted repeat followed by seven T residues that is likely to function as a factor-independent transcription terminator. The first open reading frame encodes a protein with 404 amino acid residues and a deduced molecular weight of 42,733. A search of the translated DNA sequence entries in GenBank (release 77.0) revealed that the DNA sequence in GenBank entry M98350 contained a truncated open reading frame which encoded a protein of 251 amino acids that is 48% identical with the sequence of the amino-terminal region of the NrgA protein. The DNA for this GenBank entry is reported to be of unknown bacterial origin and was isolated as a contaminant in a commercial rat liver cDNA library. This result suggests that there exists at least one other bacterium which contains a homolog of the nrgA gene. As calculated by the method of Kyte and Doolittle (21), the NrgA protein has a mean hydropathicity of 8.39, indicating that the NrgA protein is extremely hydrophobic. The hydropathicity profile of the NrgA protein is presented in Fig. 3. It is apparent that the NrgA protein contains a large number of highly hydrophobic regions that are separated by stretches of relatively hydrophilic amino acids. A hydropathicity profile with alternating hydrophobic and hydrophilic regions is typically obtained with membrane-bound proteins (21, 36). Thus, it seems reasonable to assume that the nrgA gene encodes an integral membrane protein. The NrgA protein may function as a transport protein or possibly serves as a sensor of the cell's external environment. The nrgB gene encodes a protein with 116 amino acid residues and a deduced molecular weight of 12,822. Comparison of the NrgB protein sequence with the translated GenBank sequences revealed significant sequence similarity with the E. coli glnB-encoded PI, protein and its related homologs (Fig. 4). The B. subtilis NrgB protein has the highest level of similarity with the E. coli and Klebsiella pneumoniae glnBencoded proteins (57 and 58%, respectively). In contrast, the NrgB protein is least similar to the Methanococcus thermolithotrophicus ORF105 protein (41%). To our knowledge, this is the first report of a glnB homolog in a gram-positive organism. The E. coli PI, protein is uridylyated at the tyrosine residue located at position 51 (37). The NrgB protein contains an isoleucine residue at this position, as does the Methanococcus homolog. Moreover, there are 9 contiguous amino acid residues within this region of the NrgB protein which lack similarity to the other related proteins (Fig. 4). This suggests that the B. subtilis NrgB protein may not be subject to posttranslational modification by uridylyation in the same way as the enteric PI, protein. E. coli GlnB mutants are not complemented by the B. subtilis nrgB gene (23). Identification of the nrgAB operon transcription start sites. Primer extension analysis was used to determine the transcription start sites of the nrgAB operon. With RNA isolated from cells grown in medium containing poor nitrogen sources, e.g., glutamate or proline, two major transcripts were detected (Fig. Downloaded from http://jb.asm.org/ on October 25, 2017 by guest erythromycin resistance with pNRG305. By the method described for cloning chromosomal DNA sequences adjacent to plasmid integrations (46), the plasmids pNRG323, pNRG325, and pNRG328 (Fig. 1) were isolated from SF305 chromosomal DNA digested with the restriction enzymes HindlIl, PstI, and SphI, respectively. Plasmid pNRG364 contains the spectinomycin resistance gene fronm pJL73 cloned between the MluI and SspI restriction sites located within the nrgB gene. In plasmid pNRG368, the spectinomycin resistance gene from pJL73 was cloned between the nrg-29::Tn917-lacZ insertion site in the nrg4 gene and the SspI restriction site in the nrgB gene. Plasmid pNRG365 contains the promoterless spoIL4-lacZ cat cartridge from pSGMU38 (12) cloned between the MluI and SspI restriction sites located within the nrgB gene. B. subtilis strains SF364, SF365, and SF368 were constructed by transforming strain 168 with linearized DNA of plasmids pNRG364, pNRG365, and pNRG368, respectively. The chromosomal structure of these strains was verified by Southern blot analysis (data not shown). The plasmid pSFL1 is a lacZ transcriptional fusion vector that integrates at the amyE locus. This vector is derived from the plasmid pDH32 (18), which is a promoterless derivative of ptrpBG1 (35) that has unique EcoRI and BamHI restriction sites located upstream of a spoVG-lacZ translational fusion. pSFL1 was constructed by cloning an EcoRI-SacI DNA fragment containing the E. coli trpA-lacZ translational fusion from pCED6 (11) into pDH32. pSFL1 has unique EcoRI and HindIII restriction sites located upstream of a promoterless trpA-lacZ gene. Plasmid pNRG335 was constructed by cloning the EcoRI-HindIll nrg4 promoter DNA fragment from pNRG305 into pSFL1. DNA sequencing. The nucleotide sequence was determined by the dideoxynucleotide chain-termination method (32). Sequencing reactions were performed at 70°C with Taq DNA polymerase (TaqTrack; Promega Cor.), with double-stranded plasmid DNA as the template and 2P-end-labeled oligodeoxynucleotide primers. The entire sequence was determined from both DNA strands by using the plasmids pNRG301, pNRG303, pNRG323, pNRG325, pNRG328, and pNRG342 as templates. The 290 bp of DNA sequence upstream of the nrg-29::Tn917-lacZ insertion are identical in plasmids pNRG303 and pNRG342. This indicates that the deletion of the B. subtilis chromosomal DNA present in pNRG303 did not extend into the DNA sequences reported here. The sequence of the 5-bp duplication generated by the nrg-29::Tn917-lacZ insertion was deteirmined by using synthetic oligonucleotide primers complementary to the ends of the transposon to sequences pNRG301 and pNRG303. The wild-type DNA sequence corresponding to the transposon insertion site was determined by using pNRG328 DNA as a sequencing template. No alterations in the DNA sequence adjacent to the transposon insertion junctions were observed. RNA isolation and primer extensions. RNA was isolated from B. subtilis cells grown to mid-log growth phase (70 to 90 Klett units) by extraction with guanidine thiocyanate and by CsCl centrifugation (3). Primer extension experiments were performed as previously described (15). Nucleotide sequence accession number. The nucleotide sequence reported in this communication has been assigned GenBank accession number L03216. J. BAcrERIOL. NITROGEN-REGULATED BACILLUS SUBTILIS nrgAB OPERON VOL. 176, 1994 1 111 TCGATAACATTTCTCAAACCATGTCAGGUATCTTACATGAAAATGTTTTATCATTCTTTTTTCTCTATAATGAAGAAITTATAATTGCTTTTTAT 101 -10 -35 TCTGMAGATACGGAGGAATGAGACATGCAAATGGGCGATACAGTTTTTATGTTCTTTTGCGCTTTACTCGTGTGGCTGATGACCCCGGGATTAGCGTTA nrgA M 0 M G D T V F M F F C A L L V W L M T P G L A L 201 TTTTATGGAGGAATGGTAAAGAGCAAMATGTGCTGAGCACTGCCATGCACAGTTTCTCTTCCATTGCCATCGTTTCCATCGTTTGGGTGCTGTTCGGAT F Y G G M V K S K N V L S T A M H S F S S I A. I V S I V W V L F G Y 301 ATACACTTGCCTTCGCACCAGGCMTTCMTCATCGGCGGGCTGGAGTGGGCAGGCCTCAAAGGGGTCGGATTTGATCCGGGAGATTACAGCGATACCAT 401 CCCCCACTCGTTATTTATGATGTTCCAAATGACGTTCGCCGTTCTGACTACAGCGATTATTTCCGGGGCTTTCGCAGAGCGGATGCGATTCGGC.GCTTTT P H S L F M M F Q M T F A V L T T A I I S G A F A E R M R F G A F CTTTTATTCTCGGTTTTATGGGCCTCTTTGGTTTACACACCCGTAGCGCACTGGGTATGGGGCGGCGGCTGGATCGGCCAGCTTGGAGCGCTCGATTTCG L L F S V L W A S L V Y T P V A H W V W G G G W I G Q L G A L D F A TaqI 501 F A P L A T G I N S I G G E W A G L L K G V G F P D I D Y S D T G 601 N V V H G G S S G V A G L V L A I I V L G K R K D G T A S S N H P 701 CCTCATTTACACCTTCTTAGGAGGAGCTTTGATTTGGTTCGGCTGGTTCGGCTTTMCGTCGGCAGCGCATTGACCTTAGATGGTGTGGCCATGTACGCG 801 TTCATCMCACAAACACCGCGGCTGCAGCCGGGATCGCCGGCTGGATCTTAGTAGAATGGATCATTMCAAAAMCCGACAATGCTCGGAGCGGTATCTG 901 GGGCMTCGCCGGGCTTGTCGCCATTACGCCGGCTGCCGGATTTGTCACACCGTTCGCTTCCATTATTATCGGCATCATCGGCGGAGCTGTTTGTTTCTG A I A G L V A I T P A A G F V T P F A S I I I G I I G G A V C F W 1001 GGGAGTATTCTCGCTTAAAAAGAATTCGGATACGACGACGCGCTTGACGCCTTTGGCCTGCACGGGATCGGCGGCACATGGGGCGGAATCGCMCAGGA 1101 TTATTCGCAACAACCTCTGTTMCTCAGCGGGCGCAGATGGGTTATTTTACGGTGATGCAAGCTTMTCTGGAAACAAATCGTCGCCATCGCCGCCACTT 1201 ATGTTTTTGTATTTATTGTCACTTTCGTTATTATTAAAATTGTAAGCCTCTTCCTTCCCCTTCGCGCAACTGAAGAAGAAGAGTCACTTGGGCTTGACTT 1301 AACGATGCACGGGGAAAAAGCATATCAAGATTCTATGTGAGGAGTGACGCTATGAGCGGTCAAATGTTCMGGTAGAAATTGTAACGCGTCCGGCAAATT 1401 TTGAAAAGCTGAAGCAGGAACTCGGAAAAATCGGAGTGACCTCTCTGACTTTCTCCAATGTACACGGCTGCGGCCTTCAAAAAGCACATACGGAGCTCTA I L I G V L T N F S F L F M H G T E K S I V T E Q L I A G W S A G A F V K A Y Q E K L N V I L V F G Y D D A L D A K K K T V I W F G W F G F N V G S A L T A A A A G N F A T V L G G A G D G I I K D S M K I I F G F Y G L V S L F I E W I L H G L D A S L P L L T G V T S F S N V H D G V A M Y A L K K P W K Q I L G A V T M G G T W G G I R A T nrgB M S G Q M * N L T F E G E E L Q I S V A T I V A E I E K V G C I L T G H L Y T D P A T G G A A R K A S L F N E Y L 150 1 TCGAGGGGTAAAAATAGAAAGCAATGTATACGAGCGTTTAAMAATAGAAATTGTGGTCAGCAAGGTTCCTGTTGATCMAGTGACAGAGACCGCTAAMAGG 160 1 GTGCTGAAAACGGGATCACCAGGTGACGGTAAMATATTTGTCTATGAAATCAGCAATACGATCAACATCCGCACAGGCGAAGAAGGACCTGAAGCACTTT V L K T G S P G D G K I F V Y E I S N T I N I R T G E E G P E A L* 1 701 AATATCGGTACGAGATTCGGACACTCCGGATCTCTTTTTTTGTGCACAGAATCCCCCCAGAAACCGCGATTCCTCTTCGAATTCTCTTCAAGCGCCGTTA R G V K I E N V Y S E R L K I E I V V S K V P V D Q V T E T A K R i 180 1 TTTCAGACAATCTCTATTTTTATTTGAAACTTTTCATGAGTAAGATTAGTCTACTAAATATAAAMATGTAAMAGGTGATTATTTGAACTACGAAATTTTT 1901 2001 AAAGCAATCCATGGACTATCTCATCACAATTCAGTTCTCGATTCCATTATGGTCTTCATCACGGAATATGCCATTGTCGCCTATGCCCTTATCCTATTGG CAATCTGGCTGTTTGGGAACACACAAAGCAGAAMACATGTGCTATACGCAGGCATCACAGGAATTGCAGGCCTTGTGATCAACTATTTGATTACGCTTGT 2101 TTATTTCGAACCGCGCCCGTTCGTTGCGCATACAGTGCATACACTGATTCCGCATGC FIG. 2. Nucleotide sequence of the nrgAB operon. The derived amino acid sequences of the nrg genes are shown below the coding sequences. Stop codons for the nrgA and nrgB genes are starred. The likely -10 and - 35 promoter regions are underlined once. Apparent transcription start sites at nucleotides positions 81 and 83 (see Fig. 5) (arrow), an inverted repeat upstream of the promoter (divergent arrows), and the upstream TaqI site used for cloning pNRG305 at nucleotide positions 1 through 4 are indicated. Putative ribosome-binding sites are underlined twice. The 5-bp sequence underlined at nucleotide positions 291 to 295 is the DNA target sequence duplicated by the nrg-29::Tn917-lacZ insertion. The stems of the putative transcription terminator are indicated by converging arrows at nucleotide positions 1712 to 1734. 5, lanes 1 and 3). In contrast, no extension products were observed with RNA isolated from cells grown in medium containing excess nitrogen, e.g., glutamate plus NH4' or proline plus NH4' (Fig. 5, lanes 2 and 4). The transcriptional regulation observed in the primer extension experiments is in agreement with results obtained from examination of ,B-galactosidase expression from transcriptional nrgAB-lacZ fusions (discussed in reference 2 and below). Since the 5' ends of the two transcripts are separated by only a single nucleotide, we presume that they originate from a single promoter. Examination of the DNA sequence upstream of the transcription start sites reveals the presence of an appropriately positioned sequence that is a perfect match to the B. subtilis o-Adependent -10 promoter region (26) (Fig. 2). The -35 promoter region contains only 2 nucleotides which match those in the 35 consensus sequence ITGACA (26). Many E. coli promoters with poor homology to the 35 - - consensus sequence are activated by positive regulatory proteins (30). Located immediately upstream of the nrgAB promoter is an inverted repeat (Fig. 2) which might function as the DNA binding site for such a regulatory protein. P-Galactosidase expression of lacZ fusions. A 295-bp TaqI DNA fragment containing 80 bp of DNA upstream of th,e nrgAB transcriptional start sites was transcriptionally fused to the lacZ gene [(nrgA-lacZ)335] and integrated as a single copy at the amyE locus in the B. subtilis chromosome. Regulation of expression of this lacZ fusion was examined by growing SF335 cells in medium containing excess or limiting nitrogen (Table 2). 1-Galactosidase levels were 8,800-fold higher in extracts of cells grown with a poor nitrogen source, glutamate, -than in extracts of cells grown in medium with excess nitrogen, glutamate plus NH44. Since the regulation of the [(nrgA-lacZ)335] fusion duplicates the regulation of the nrg-29::Tn917-lacZ fusion (2), all of the cis-acting sites required for the regulation Downloaded from http://jb.asm.org/ on October 25, 2017 by guest F F Y T 112 J. BACTERIOL. WRAY ET AL. 1 2 s"- A C G T 3 4 x CL .2 V "2 I1 @extension. A 32P-end-labeled oligodeoxynucleotide primer comple- I t Z J X .......................... ...... g FIG. 5. Identification of the 5' termini of nrgAB RNA by primer n a y t u l o i e 6 to o 1 a s d f r t e p i e 160 180 iin Fi Fig..,22 was used for the primer to nucleotides vW.,..............m ^ ~~~~~~~~mentary } extension analysis (lanes 1 to 4) and for dideoxy sequencing of pNRG305 (lanes A, C, G, and T). RNA was isolated from B. subtilis | .... | ., | * | W minimal s | g | medium containing various nitrogen in glucose ~168 grown S S 241 sources. Lanes: 1, glutamate; 2, glutamate plus 3, proline; 4, t il l ,s -2@1 I - IS w f * | W | * ....... NH4+; proline plus NH4+. FIG. 3. Hydropathy profile of the NrgA protein. A version of the SOAP program (PC/GENE; Intelligenetics, Inc.) described by Kyte and Doolittle (21) was used to determine the average hydropathy of a moving segment of 15 amino acid residues. The dotted line at -5 is the standard midpoint line that is used to divide the hydrophobic domains of the protein (above the line) from the hydrophilic. domains (below the line). and expression of the nrgAlB operon are probably contained within the 295-bp TaqI DNA fragment. In addition, the regulation of the [(nrgA-LacZ)335] fusion is not altered in strains containing deletions of either the nrgB gene or the operon (Table 2). This suggests that the NrgA and NrgB nr&4B gene products are not required for the nitrogen-regulated expression of the nrgAB operon. To determine whether the expression of the nrgB gene is UMP BS Ec R4 C GLQKAHTELYRGVKI E S N V Y EGIV TSLFS VE1 E N K K I D A I I K P F K L D D V R E A L A E V G I T G M T V T E V K G F G R Q K G H T E L Y R G A E Y M VD F L NSGQ N Kp MKK I DAI I KPFKLDDVREALAEVGI TGMTVTEVKGFGR KG HTELYRGAEYMVD FL Ap NK K 0 I EAI I KP F KLD E VKEAL EVG I K G I T V T E A K G F G RQ K G H T E L Y R G A E Y V V D F L RI E|A|K G F G R O K G H T D L YR G AE Y I V D F L SLSV M K K I E A I I K P f K L D E V R -S P S G V G L Q G I T V T E AK G F G R O K G H T E L Y R G A E Y V V D F L Rc M K K V E AI BJ G L QG I T V T K K I EA I I K P F K L D E V R Sy AG QG L SVE V K G F G R Q KG H T E L YR G A EY VD NK K I E A I I R P F K L D E V K I A L V A G I V GMTVS E VR G f G R Q K G R RG E YVE Mt NN Bs E I K P F K L D E VK EAL N AGYP AFF RKIRDKVDDVDSE V EIVVSKVP K I E I V VVD T AKRVLK a D I V D T C V D T|I I|R T KINSB GQ L---- GDGKI F;VYEI FL F L FYd GE IR TGEE I R T G E ED D A I Ec PK Kp P K VI K I E I V V T D D I V D T C V D TJ I I t TA Q T G K I G D G K I F V F D V A RV I R I R T G E E|D D A A I| Az |P K V K I EV V Bj P K V K I E I V T G K I G D G K I F V F D VA R Rt SDEL V E R A It E A IIHCA AB T G R I G D G K I F V TIPIV E E V V R I R TG E K D A I G|D D L V E|R A I D A I Rft A A Q T B R I G D G K I F V S N!I E El R I R T G E|S|G L D A! P It V K V E V V L|A|D E ;E AS I E A I R K A A Q T G R I G D G K I F V S NIV E E V I R I R T G E T G I D A 1| Rc P K V K I E V L P DE N Sy QK L K L E I V V E D| Q V D T V I D 0 E A I VGA ARTEKI G D G K I F V S|S|I EQA 1 V A^I^ |R T G|E|I G D G K I F V S P|V D R I R T G E T IR T[qED A Vl R T G E|K N A D A 1I| FIG. 4. Alignment of the deduced amino acid sequence of the B. subd&E NrgB protein with similar protein sequences from other bacteria. Amino acid residues that are identical or similar among the nine proteins are boxed. Groups of similar amino acids are LVI, AG, ST, DE, and RK The position of the tyrosine residue which is uridylyated in E. coli is indicated by the arrow marked UMP. Abbreviations and sequence references: Bs, B. subtilis; Ec, E. coli (37, 43); Kp, K pneumoniae (19); Az, Azospirillum brasilense (10); Bj, Bradyrhizbium japonicum (24); RI, Rhizobium leguminosarum (7); Rc, Rhodobacter capsulatus (20), Sy, Synechococcus strain PCC 7942 (41); Mt, M. thermolithotrophicus ORF105 (39). Downloaded from http://jb.asm.org/ on October 25, 2017 by guest Amino Acid Residue VOL. 176, 1994 NITROGEN-REGULATED BACILLUS SUBTILIS nrgAB OPERON TABLE 2. 1-Galactosidase levels in nrg-lacZ fusion strains Strain Relevant genotype SF335 SF344 AamyE::FD(nrgA-lacZ)335 AamyE::A(nrgA-lacZ)335 SF348 AamyE::I(nrgA-lacZ)335 ,-Galactosidase sp act (U/mg of protein) by nitrogen source" Glutamate + NH4 Glutamate 0.02 0.01 177 160 <0.02 172 AnrgB364::spec AnrgAB368::spec SF365 1'(nrgB-lacZ)365 <0.02 13.4 aAverage of three to four determinations. The values did not vary by more than 20%. ,B-Galactosidase activity was corrected for endogenous 3-galactosidase activity present in strain SF330, e.g., 0.08 in glutamate-plus-NH4Cl-grown cells and 0.26 in glutamate-grown cells. Cells were grown in MOPS minimal medium containing 0.5% glucose as the carbon source and the indicated nitrogen sources. ACKNOWLEDGMENTS We thank Patricia Rice and Florence Pettengill for their technical assistance, W. Hillen for providing pDH32, J. Le Deaux for pJL73, D. A. Morrison for pJDC9, J. Mueller for pSGMU38, A. L. Sonenshein for pCED6, and P. Youngman for pTV20, pTV21A21 and KE93. We are grateful to B. Magasanik for helpful discussions. This work was supported by Public Health Service research grant RO1-AI23168 from the National Institutes of Health. REFERENCES 1. Anagnostopoulos, C., P. J. Piggot, and J. A. Hoch. 1993. The genetic map of Bacillus subtilis, p. 425-461. In A. L. Sonenshein, J. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, D.C. 2. Atkinson, M. R., and S. H. Fisher. 1991. Identification of genes and gene products whose expression is activated during nitrogenlimited growth in Bacillus subtilis. J. Bacteriol. 173:23-27. 3. Atkinson, M. R., L. V. Wray, Jr., and S. H. Fisher. 1990. Regulation of histidine and proline degradation enzymes by amino acid availability in Bacillus subtilis. J. Bacteriol. 172:4758-4765. 4. Chambers, S. P., S. E. Proir, D. A. Barstow, and N. P. Minton. 1988. The pMTL nic cloning vectors. I. Improved pUC polylinker regions facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139-149. 5. Chasin, L. A., and B. Magasanik. 1968. Induction and repression of the histidine-degrading enzymes of Bacillus subtilis. J. Biol. Chem. 243:5165-5178. 6. Chen, J., and D. A. Morrison. 1987. Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene 55:179-187. 7. Colonna-Romano, S., A. Riccio, M. Guida, R. Defez, A. Lamberti, M. laccarino, W. Arnold, U. Priefer, and A. Puhier. 1987. Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum. Nucleic Acids Res. 15:1951-1964. 8. Debarbouille, M., I. Martin-Verstraete, F. Kunst, and G. Rapoport. 1991. The Bacillus subtilis sigL gene encodes an equivalent of u" from gram-negative bacteria. Proc. Natl. Acad. Sci. USA 88:9092-9096. 9. Deuel, T. F., and E. R. Stadtman. 1970. Some kinetic properties of Bacillus subtilis glutamine synthetase. J. Biol. Chem. 245:52065213. 10. de Zamaroczy, M., F. Delorme, and C. Elmerich. 1990. Characterization of three different nitrogen-regulated promoter regions for the expression of glnB and glnA in Azospirillum brasilense. Mol. Gen. Genet. 224:421-430. 11. Donnelly, C. E., and A. L. Sonenshein. 1984. Promoter-probe plasmid for Bacillus subtilis. J. Bacteriol. 157:965-967. 12. Errington, J. 1986. A general method for fusion of the Escherichia coli lacZ gene to chromosomal genes in Bacillus subtilis. J. Gen. Microbiol. 132:2953-2966. 13. Fisher, S. H., and A. L. Sonenshein. 1984. Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression. J. Bacteriol. 157:612-621. 14. Fisher, S. H., and A. L. Sonenshein. 1991. Control of carbon and nitrogen metabolism in Bacillus subtilis. Annu. Rev. Microbiol. 45:107-135. 15. Fisher, S. H., and L. V. Wray, Jr. 1989. Regulation of glutamine synthetase in Streptomyces coelicolor. J. Bacteriol. 171:2378-2383. 16. Golden, K. J., and R. W. Bernhohr. 1985. Nitrogen catabolite repression of the L-asparaginase of Bacillus licheniformis. J. Bacteriol. 164:938-940. 17. Gough, J. A., and N. E. Murray. 1983. Sequence diversity among related genes for recognition of specific targets in DNA molecules. J. Mol. Biol. 166:1-19. 18. Henner, D. J. (Genentech). 1991. Personal communication. 19. Holtel, A., and M. Merrick. 1988. Identification of the Klebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles. Mol. Gen. Genet. 215:134-138. 20. Kranz, R. G., V. M. Pace, and I. M. Caldicott. 1990. Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus. J. Bacteriol. 172:53-62. 21. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157:105-132. 22. Lopilato, J., S. Bortner, and J. Beckwith. 1986. Mutations in a new Downloaded from http://jb.asm.org/ on October 25, 2017 by guest nitrogen regulated, a portion of the nrgB gene was replaced with a promoterless lacZ cat cassette, and the resulting nrgBlacZ fusion was used to replace the chromosomal nrgB gene (SF365 in Fig. 1). 1-Galactosidase levels in SF365 cells grown with excess and limiting nitrogen were determined (Table 2). f-Galactosidase synthesis was derepressed in cells grown with glutamate as a nitrogen source, while only background levels of 3-galactosidase activity were detected in cells grown with glutamate and NH4' as a nitrogen source. Thus, expression of the nrgB gene is nitrogen regulated. Since expression of ,B-galactosidase from the chromosomal nrgB-lacZ fusion and transcription from the promoter located upstream of the nrgA gene show similar patterns of regulation, there is no constitutive promoter which transcribes the nrgB gene. Interestingly, expression of the E. coli glnB gene is not regulated in response to nitrogen availability (42). Growth phenotype of strains containing AnrgAB and AnrgB mutations. Since the B. subtilis GS protein is not known to be posttranslationally modified (9, 13), the NrgB "PI,-like" protein is unlikely to regulate GS activity in B. subtilis. Indeed, GS specific activity in permeabilized SF12 (nrg-29::Tn917-lacZ) cells grown in glucose minimal medium containing either glutamate plus NH4' or glutamate alone as the nitrogen source was similar to that seen in SFiO (wild-type) cells (data not shown). To investigate the roles played by the NrgA and NrgB gene products in nitrogen metabolism in B. subtilis, strains SF364 (AnrgB) and SF368 (zXnrgAB) (Fig. 1) were constructed. Strains SF364 (AnrgB) and SF368 (AnrgAB) sporulated at wild-type frequencies in either Difco sporulation medium or SterliniMandelstam resuspension medium (data not shown). By streaking the wild-type and nrgAB deletion strains on glucose minimal plates containing different nitrogen sources (allantoin, y-aminobutyrate, L-asparagine, glucosamine, L-glutamate, Lhistidine, L-isoleucine, KNO3, L-ornithine, L-threonine, urea, and L-valine), we determined that strains SF364 (AnrgB) and SF368 (AnrgAB) are not defective in the utilization of these nitrogen sources. However, on glucose-KNO3 minimal plates, both deletion strains grew more slowly than did the wild-type strain, although all three strains eventually formed colonies with similar sizes. This suggests that the NrgA and NrgB gene products either participate in nitrate utilization or facilitate adaptation to growth on this medium. 113 114 23. 24. 25. 26. 27. 29. 30. 31. 32. 33. 34. chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol. Gen. Genet. 205:285-290. Magasanik, B. (Massachusetts Institute of Technology). 1992. Personal communication. 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