Gene organisation and regulatory sequences in the sucrose utilisation cluster of Bacillus stearothermaphilus NUB36

Gene 195 (1997) 195–200

Gene organisation and regulatory sequences in the sucrose utilisation cluster of Bacillus stearothermaphilus NUB36 Yang Li, Thomas Ferenci * Department of Microbiology G08, University of Sydney, Sydney, NSW 2006, Australia Received 31 October 1996; received in revised form 27 January 1997; accepted 29 January 1997; Received by M. Salas

Abstract The nucleotide sequence of the surP and surT genes in a sucrose-utilisation cluster cloned from Bacillus stearothermophilus NUB36 was determined. The surP gene encoded a protein of 466 amino acid residues and shared 60–62% amino acid identity with the sucrose-specific enzyme II components of the phosphotransferase system of Bacillus subtilis, Salmonella typhimurium and Klebsiella pneumoniae. SurP, like other sucrose EIIs, lacked the hydrophilic domain containing the first (IIA) phosphorylation site. The surT gene encoded a 278 amino acid polypeptide which showed 63.1% and 54% amino acid identity to the B. subtilis antiterminators SacT and SacY, respectively. A region containing a palindromic structure preceding surP was highly homologous to the regulatory transcription termination regions of the sacPA and sacB operons of B. subtilis and the bgl operon of Escherichia coli. Hence the sucrose gene cluster of B. stearothermophilus NUB36 is very similar to the B. subtilis sacPA operon in terms of gene order and regulatory organisation. © 1997 Elsevier Science B.V. Keywords: Phosphotransterase system; Transcription termination; Antitermination; Sucrose phosphate hydrolase

1. Introduction Nothing has been reported about the organisation of sucrose genes and the regulation of sucrose utilisation from thermophilic organisms. In most bacteria, the initiation of sucrose metabolism involves transport and phosphorylation using the phosphoenolpyruvate: sugar phosphotransferase system (PTS ) for sucrose uptake (Postma et al., 1993; Titgemeyer et al., 1996). In Bacillus subtilis, the sucrose-specific component of the PTS (SacP) is encoded by a gene in the sacPA operon, which also contains the structural gene for the sucrose–sucrose phosphate hydrolase ( Klier and Rapoport, 1988). We report here that Bacillus stearothermophilus has a very similar organisation of the genes involved in sucrose utilisation. Regulation of sucrose genes has been extensively studied in B. subtilis by both genetic and molecular analysis ( Klier and Rapoport, 1988; Debarbouille et al.,

* Corresponding author. Tel. +61 2 93514277; Fax: +61 2 93514571; e-mail: t.ferenci@ microbio.su.oz.au 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 39 - X

1991). Mutants causing constitutive expression of EII Scr and sucrose hydrolase were selected and their analysis led to the identification of the loci involved in sacPA regulation (Lepesant et al., 1972). The sacTencoded protein of B. subtilis is a transcription antiterminator (Arnaud et al., 1996) and shares strong homology with the antiterminators of both B. subtilis SacY and the E. coli BglG (Debarbouille et al., 1990). sacR was found to contain a long palindromic structure which consists of two 31 bp stretches located upstream of the sacB gene and shares extremely high homology with the palindromic structures involved in transcription termination/antitermination upstream of the B. subtilis sacB gene (Crutz et al., 1990) and the E. coli bglG gene (Schnetz and Rak, 1988). In contrast, the sucrose EIIs from enteric bacteria are regulated by a repressor (Jahreis and Lengeler, 1993). Evidence is presented that the DNA sequence of B. stearothermophilus NUB36 contains an antiterminator (surT) sequence as well as a regulatory region (surR) containing a palindromic structure. The organisation of both the structural and regulatory sequences in the thermophile show a high similarity to those involved in sucrose utilisation of B. subtilis.

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2. Experimental 2.1. Sequence of the sucrose cluster from B. stearothermophilus A previously described clone, containing 4.5 kb DNA from B. stearothermophilus NUB36, conferred on E. coli the ability to utilise sucrose (Li and Ferenci, 1996). The complete nucleotide (nt) sequence of this clone was obtained. In addition to the sucrose hydrolase (SurA) essential for growth of E. coli on sucrose (Li and Ferenci, 1996), analysis of the remaining 3 kb DNA revealed two complete open reading frames (ORF1; ORF2) leading into the 5∞ end of the surA gene. The ORF1 and ORF2 were separated by 183 bp (nt 1177–1359) and the ORF2 and surA were separated by 152 bp (nt 2758–2909). A palindromic structure was located between ORF1 and ORF2 (nt 1248–1359). The ORF1 and ORF2 were designated surT and surP, respectively because of their high sequence homology with B. subtilis sacT and sacP, respectively. Analysis of the nt sequence in Fig. 1 revealed that surT consisted of 834 bp encoding a 277 amino acid protein with a calculated molecular weight of 32 016 and surP contained 1338 bp encoding a 465 amino acid protein with a calculated molecular weight of 49 599. surT most likely starts at nt 343, preceded by a translational start signal GGGA at a distance of 4 bp, suggesting that the surT gene starts with the ATG codon at this site. The surT gene ended at a TGA (nt 1176). surP is postulated to start at an ATG (nt 1360) and end at a TAG (nt 2759). Here again, the initiator codon is preceded by a translational start signal, GAGA, at a distance of 11 bases, suggesting that it is the beginning of gene surP. Further sequence analysis of the upstream regions of each ORF revealed two potential promoter sites (assessed according to the consensus promoter of B. stearothermophilus, TTGAAt/c at −35 and TATTa/cT in the −10 region, ( Wu and Welker, 1991) in the upstream regions of surT and surP. The first promoter site was located 20 bp upstream of the start codon ATG of surT (Fig. 1). The −10 and −35 regions were separated by 17 bp, and four of the six nts were identical when compared with the consensus promoter sequence of B. stearothermophilus. The second promoter site was located 132 bp upstream of the start codon of surP. 17 nts were found between the −10 and −35 regions, and three out of six nts are identical with the B. stearothermophilus −35 consensus sequence and four out of six with −10 sequence. No potential promoter site was found upstream of the downstream surA gene, hence, it is possible that surP and surA share the same promoter. There is a palindromic structure upstream of surP, and it shares strong homology with the reported palindromic structures involved in the regulation of sugar utilisation (Schnetz and Rak, 1988).

2.2. Comparison of B. stearothermophilus NUB36 SurP with other proteins The deduced amino acid sequence of SurP from B. stearothermophilus NUB36 shared about 62% identity with the B. subtilis SacP protein and 60% identity with other sucrose-specific EIIs of the PTS system in S. typhimurium and K. pneumoniae, as shown in the alignment in Fig. 2. The enzymes II Scr from enteric bacteria and B. subtilis require a separate IIA (using glucose IIAGlc when cloned in E. coli) for activity. On the basis of sequence alignments, EIIs may be grouped into at least four classes (Postma et al., 1993) and the sequences of EIIs specific for sucrose and b-glucosides are more closely related to each other than to other members of the class ( Titgemeyer et al., 1996). The hydrophilic NH -terminal region of SurP up to residue 110 corres2 ponds to a typical IIB domain. As shown in the alignment in Fig. 2, the amino acid residues surrounding C26, the likely phosphorylation site (Meins et al., 1988) of SurP (sites 15–31), were highly conserved [EII consensus sequence: AHCATRLR, (Lengeler et al., 1990)]. The highly conserved GITE motif (Lengeler et al., 1990) was also found at position 370–373 (Fig. 2). A histidine residue located at position 310 of SurP was identical in all the compared enzymes II sequences (Fig. 2), and the amino acid residues around position 310 showed high similarity with the proposed phosphorylation site 2 of the plasmid-encoded sucrose enzyme II from Salmonella typhimurium (Saier et al., 1988). In total, 73 of the 77 totally conserved residues identified by Lengeler et al. ( Titgemeyer et al., 1996) in sucrose EIIs were found in SurP.

2.3. Comparison of B. stearothermophilus NUB36 SurT with other proteins The putative surT-encoded protein showed high amino acid identity to the antiterminators SacT and SacY of B. subtilis (63% identity in 249 amino acids overlap and 54% identity in 276 amino acids overlap, respectively). Both antiterminators, SacT and SacY, are involved in the regulation of sucrose metabolism of B. subtilis (Debarbouille et al., 1990; Zukowski et al., 1990). The surT-encoded protein also showed strong similarity to the regulatory protein BglG of E. coli (37.8% identity in 278 amino acids overlap (Schnetz and Rak, 1988). Six highly conserved areas were observed when the surT encoded protein sequence was compared with the alignment of other antiterminator sequences ( Figs. 3A–F ). The identical and conserved amino acid residues were spread evenly throughout the whole protein sequences except the NH -termini. The likely targets 2 (palindromic termination structures) of these antitermi-

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Fig. 1. The DNA sequence of the sucrose cluster of the B. stearothermophilus NUB36 chromosome. The B. stearothermophilus DNA clone obtained previously (in plasmid pAM2001, Li and Ferenci, 1996) was subcloned into pGEM-7Zf(+) vector (Promega, Madison, WI ) or pT7T3 (Pharmacia: AMRAD Pharmacia Biotech, Victoria, Australia) using strains previously described (Li and Ferenci, 1996). Nested deletions were obtained by the method of Henikoff (Henikoff, 1984). Nt sequences were determined by the dideoxy-chain termination method with DNA templates from singlestranded phagemid DNA or double-stranded plasmid DNA (Del Sal et al., 1988). Sequencing used the pUC/M13 17-mer universal forward sequencing primer or the reverse sequencing primer, using Taq DNA polymerase (Promega) and fluorescent- dye-coupled primers and a model ABI37OA automated DNA sequencer. Sequencing was performed for the entire length of both strands. The likely Shine–Dalgarno sequence (SD), start codons, termination codons and ‘‘−10’’ ‘‘−35’’ regions are underlined. Arrows indicate the palindromic sequences. The beginning of each orf is labelled under the start codon. The nt sequences have been submitted to Genbank under accession numbers U34872 (surA) U34873 (surT) and U34876 (surP).

nators also shared strong similarity (Fig. 4, boxes A and B). 2.4. Comparison of the palindromic structure preceding surP with the palindromic structures involved in the regulation of sugar metabolism In B. subtilis, two palindromic structures (t1 and t2) were found to be important in the regulation of sucrose

metabolism (Debarbouille et al., 1990; Zukowski et al., 1990). The two palindromic structures (t1 and t2) share strong homology in two regions designated as box A and box B (Fig. 4). Similar palindromic structures (f1 and f2) regulated by antiterminators have been discovered in E. coli (Schnetz and Rak, 1988) and were involved in the regulation of aryl-b-glucoside utilisation. The conserved sequences were called ribonucleic antiterminators (RATs) by Aymerich and Steinmetz (1992),

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Fig. 2. Comparison of the protein encoded by surP with other enzymes II of the PTS system. (*) indicates a perfect match across all sequences and (.) shows conservative substitutions. SurP sequence is from B. stearothermophilus NUB36, BssacP is enzyme II encoded by sacP gene of B. subtilis, BssacX is enzyme II encoded by sacX of B. subtilis, and KpscrA is enzyme II encoded by scrA of K. pneumoniae. The conserved C26 and H309 residues are in bold type and the enzyme II motifs HCATRLR and GITE are underlined.

who showed these to be the specificity determinants recognised by antiterminator proteins. The palindromic structure (surR) preceding surP of B. stearothermophilus NUB36 was compared with t1, t2 and f1, f2 and showed significant similarity in both the box A and box B regions ( Fig. 4). In the box A region, 9 out of 17 nts were identical in all sequences. In the box B region, 13/17 nts were identical and all 17 nt in B. stearothermophilus were identical to the t1, t2 sequences of B. subtilis. These two conserved regions have not been found in ordinary termination structures; thus it is likely

that the box A and box B regions in B. stearothermophilus NUB36 also contain the RAT site. 2.5. Conclusions The sequence of the sucrose gene cluster from B. stearothermophilus NUB36 shows a similar order of genes and regulatory sites as the B. subtilis sacPA operon. With the high level of homology to the published palindromic structures, especially in the proposed conserved functional regions (RAT region,

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Fig. 3. Comparison of the amino acid sequence of SurT with transcription antiterminator proteins. (*) indicates a perfect match across all sequences and (.) shows conservative substitutions. SurT is the protein encoded by orf1 of B. stearothermophilus NUB36, SacT represents the sacT-encoded antiterminator of B. subtilis, SacY is the antiterminator encoded by sacY of B. subtilis and BglG is the positive regulatory protein encoded by bglG of E. coli.

Fig. 4. Comparison of the palindromic structure from B. stearothermophilus NUB36 (surR) with the t1 (sacpA) and t2 (sacB) palindromic structures from B. subtilis (Debarbouille et al., 1990; Aymerich and Steinmetz, 1992), and the f1 and f2 palindromic structures from E. coli bgIF and bgIG (Schnetz and Rak, 1988). The RAT region corresponds to the ribonucleic acid antiterminator target region defined by Aymerich and Steinmetz (1992) and the terminator stem-loop is shown by the dashed lines.

Fig. 4), it is likely that sucrose genes in B. stearothermophilus NUB36 are regulated by the same mechanism as in B. subtilis. The RAT region in surR is the likely target of the SurT antiterminator, but this remains to be demonstrated. The amino acid sequences of SurT, SurP reported here and SurA (Li and Ferenci, 1996) have 55–63% identity to the corresponding proteins from B. subtilis, so are also expected to have similar functional roles.

References Arnaud, M., Debarbouille, M., Rapopon, G., Saier, M.H., Reizer, J., 1996. In vitro reconstitution of transcriptional antitermi nation by the SacT and SacY proteins of Bacillus subtilis. J. Biol. Chem. 271, 18966–18972. Aymerich, S., Steinmetz, M., 1992. Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BgIG/SacY family. Proc. Natl. Acad. Sci. USA 89, 10410–10414.

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Crutz, A.M., Steinmetz, M., Aymerich, S., Richter, R., Le, C.D., 1990. Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system. J. Bacteriol. 172, 1043–1050. Debarbouille, M., Arnaud, M., Fouet, A., Klier, A., Rapoport, G., 1990. The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators J. Bacteriol. 172, 3966–3973. Debarbouille, M., Martin-Verstraete, I., Arnaud, M., Klier, A., Rapoport, G., 1991. Positive and negative regulation controlling expression of the sac genes in Bacillus subtilis . Res. Microbiol. 142, 757–764. Del Sal, G., Manfioletti, G., Schneider, C., 1988. A one-tube plasmid mini-preparation suitable for sequencing. Nucleic Acids Res. 16, 9878 Henikoff, S., 1984. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351–359. Jahreis, K., Lengeler, J.W., 1993. Molecular analysis of two ScrR repressors and of a ScrR–FruR hybrid repressor for sucrose and fructose specific regulons from enteric bacteria. Mol. Microbiol. 9, 195–209. Klier, A.F., Rapoport, G., 1988. Genetics and regulation of carbohydrate catabolism in Bacillus . Annu. Rev. Microbiol. 42, 65–95. Lengeler, J.W., Titgemeyer, F., Vogler, A.P., Wohrl, B.M., 1990. Structures and homologies of carbohydrate: phosphotransferase system (PTS) proteins. Philos. Transact. R. Soc. London B 326, 489–504. Lepesant, J.A., Kunst, F., Lepesant-Kejzlarova, J., Dedonder, R., 1972. Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg. Mol. Gen. Genet. 118, 135–160. Li, Y., Ferenci, T., 1996. The Bacillus stearothermophilus NUB36 surA

gene encodes a thermophilic sucrase related to Bacillus subtilis Hal. Microbiology 142, 1651–1657. Meins, M., Zanolari, B., Rosenbusch, J.P., Erni, B., 1988. Glucose permease of Escherichia coli. Purification of the IIGlc subunit and functional characterization of its oligomeric forms. J. Biol. Chem. 263, 12986–12993. Postma, P.W., Lengeler, J.W., Jacobson, G.R., 1993. Phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev. 57, 543–594. Saier Jr., M.H., Yamada, M., Erni, B., Suda, K., Lengeler, J., Ebner, R., Argos, P., Rak, B., Schnetz, K., Lee, C.A., Stewart, G.C., Breidt Jr., F., Waygood, E.B., Peri, K.G., Doolittle, R.F., 1988. Sugar permeases of the bacterial phosphoenolpyruvate-dependent phosphotransferase system: sequence comparisons. FASEB J. 2, 199–208. Schnetz, K., Rak, B., 1988. Regulation of the bgl operon of Escherichia coli by transcriptional antitermination. EMBO J. 7, 3271–3277. Titgemeyer, F., Jahreis, K., Ebner, R., Lengeler, J.W., Molecular analysis of the scrA and scrB genes from Klebsiella pneumoniae and plasmid pUR4OO which encode the sucrose transport protein enzyme IIScr of the phosphotransferase system and a sucrose6-phosphate invertase. 1996. Mol. Gen. Genet. 250, 197–206. Wu, L., Welker, N.E., 1991. Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription. J. Bacteriol. 173, 4877–4888. Zukowski, M.M., Miller, L., Cogswell, P., Chen, K., Aymerich, S., Steinmetz, M., 1990. Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory peptides. Gene 90, 153–155.