FEMS Microbiology Letters 173 (1999) 217^222

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1 FEMS Microbiology Letters 173 (1999) 217^222 Expression of the genes for guanyl-speci c ribonucleases from Bacillus intermedius and Bacillus pumilus is regulated by the two component signal transduction system PhoP-PhoR in B. subtilis Lilia V. Znamenskaya a; *, Olga A. Vershinina a, Valentina I. Vershinina a, Inna B. Leshchinskaya a, Robert W. Hartley b a Laboratory of the Biosynthesis and Bioengineering of Enzymes, Department of Microbiology, Kazan State University, Kazan , Russia b Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Abstract Received 2 December 1998; received in revised form 2 February 1999; accepted 3 February 1999 Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and B. pumilus (Rnase Bp) in phosphate deficient conditions, contain regions similar to appropriate consensus sequences in promoters of the PHO regulated genes of B. subtilis. A number of genes expressed in response to phosphate starvation in B. subtilis are regulated by the two component signal transduction system PhoP-PhoR. Expression of recombinant genes for binase and RNase Bp in B. subtilis strains with mutations in the regulatory protein genes of the PHO regulon was studied. Their expression is strongly regulated by the regulatory proteins of the B. subtilis PHO regulon. z 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Ribonuclease; Bacillus; Gene regulation; PHO regulon 1. Introduction Bacteria of the genus Bacillus respond to the depletion of nutrients by a set of di erent reactions, including the production of degradative enzymes [1,2]. Secreted guanyl-speci c ribonucleases are synthesized by many Bacillus species: Bacillus amyloliquefaciens, Bacillus intermedius, Bacillus pumilus, Bacillus thuringiensis, Bacillus coagulans, Bacillus circulans. These enzymes catalyze the cleavage of * Corresponding author. Tel.: +7 (8432) ; Fax: +7 (8432) ; lilia.znamenskaya@ksu.ru phosphodiester bonds in RNA, oligo and polynucleotides into mono and small oligonucleotides by a two stage mechanism with the formation of 2P-3Pcyclic phosphates with later hydrolysis to the nucleoside-3p-phosphates [3]. The genes for guanyl-speci c ribonucleases have homologous parts encoding mature proteins. Guanyl-speci c ribonucleases from B. intermedius (binase) and B. pumilus (RNase Bp) are synthesized during the transition from exponential growth to the stationary phase as a result of inorganic phosphate (P i ) depletion [4]. A number of B. subtilis genes, induced in response to phosphate deprivation, depend / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S (99)

2 218 L.V. Znamenskaya et al. / FEMS Microbiology Letters 173 (1999) 217^222 on PhoP and PhoR proteins for their expression and are identi ed as PHO regulon genes. PhoP and PhoR form a two component signal transduction system, which regulates the phosphate response in B. subtilis through a cascade of protein phosphorylation. Histidine kinase PhoR receives an extracellular signal and transmits it to the response regulator PhoP, which, in PhoPVP form, is directly involved in the activation of PHO regulon genes [2,5]. The traditional reporter protein for PHO regulation in prokaryotes is alkaline phosphatase. Ribonucleases precede phosphatases in the reactions of disengaging phosphate from RNA molecules. This led us to a proposal that the genes for secreted ribonucleases and phosphatases of bacilli might be controlled by the same mechanism. The experiments described in this paper were designed to determine whether the regulation of the expression of recombinant binase and RNase Bp genes is controlled by PhoP-PhoR proteins in B. subtilis host strains. Barnase is a homologous ribonuclease from B. amyloliquefaciens. Barstar is its speci c inhibitor and expression of the latter is necessary to protect a host bacterium from the toxic e ect of any of these ribonucleases [3]. 2. Materials and methods 2.1. Bacterial strains and plasmids B. subtilis JH642 (phea1 trpc2), MH5117 (phea1 trpc2 phopv EcoRI mdh: :Tet r ), MH5124 (phea1 trpc2 phorv BalI: :Tet r ) strains were used. Strains were kindly provided by Dr Hulett. Plasmids used in this study are listed in Table 1. The recombinant circle PCR technique of Jones and Howard [6] was used to recombine portions of pml5 [14] and pml163 [7] to make pbi and like portions of pml61 [4] and pml253 [14] to make pbp, yielding cassettes with the binase or Rnase Bp gene, each on its own promoter and signal sequence, and with barstar on its own promoter. Transformed clones were selected by hybridization with appropriate 32 P-labelled oligonucleotides. RNase production was tested on nutrient agar plates with yeast RNA in a nal concentration of 5 g l 31 [8]. For expression in B. subtilis, the Escherichia coli plasmids were joined with pub110 at the unique EcoR1 site of each plasmid to make the shuttle plasmids pmz55 and pmz56 for binase and Rnase Bp, respectively. pmt420 was a similar construct of the entire barnase gene in the same vector except that the E. coli plasmid was combined with pc194 to make the shuttle. Plasmid isolation and transformation of competent B. subtilis cells with plasmid DNA was carried out by a standard method [9] DNA sequencing Binase and RNase Bp genes from pbi and pbp were sequenced by the dideoxy chain termination method of [10], using Sequenase Version 2.0 (USB) and the appropriate primers. Table 1 List of plasmids: RNase-barstar cassette Plasmid Antibiotic resistance RNase-barstar cassette RNase promoter Signal sequence Structural gene Barstar promoter pml5 Ap R Binase Binase Binase tac pml61 Ap R RNase Bp RNase Bp RNase Bp tac pml163 Ap R tac phoa Binase Barstar pml253 Ap R tac phoa RNase bp Barstar pbi Ap R Binase Binase Binase Barstar pbp Ap R RNase Bp RNase Bp RNase Bp Barstar pmz55 Ap R,Km R Binase Binase Binase Barstar pmz56 Ap R,Km R RNase Bp RNase Bp RNase Bp Barstar pmt420 Ap R,Cm R Barnase Barnase Barnase Barstar

3 L.V. Znamenskaya et al. / FEMS Microbiology Letters 173 (1999) 217^ Growth conditions B. subtilis strains were grown at 30³C in a complex phosphate de cient medium containing: low phosphate peptone 2.0%, glucose 1.0%, CaCl %, MgSO 4 W7H 2 O 0.03%, NaCl 0.3%, MnSO %, ph 8.5. The content of P i in low phosphate peptone does not exceed 0.4 mg g 31 of dry substance Enzyme assay The RNase assay was carried out by the modi ed acid precipitation method of [11], based on estimating the amount of acid soluble, low molecular mass nucleotide fragments, produced by ribonuclease from hydrolysis of highly polymerized RNA. One unit of activity was de ned as the amount of the enzyme which catalyses an increase in A 260 of 1.0 under the above conditions: opt. u (ml h) 31. The speci c activity was calculated by dividing the RNase activity as assayed by the A 590 of the culture. 3. Results We have previously shown that, under conditions of phosphate de ciency, binase and RNase Bp are synthesized by B. intermedius 7P and B. pumilus KMM62 after the exponential growth [4]. Nucleotide sequences of the promoters of binase and Rnase Bp are compared in Fig. 1 with promoters of the B. subtilis PHO regulon genes. Promoters of binase and RNase Bp are closely related, di ering by only two nucleotides in the sequences shown. They have consensus sequences in the 310 (6 of 6 bp) and 335 (5 of 6 bp) regions necessary for c A RNA polymerase interaction. Also, the binase and RNase Bp promoters contain two regions homologous to the TTAACA conserved sequence which has been observed in the promoters of PHO regulon genes of B. subtilis [12]. One of them is situated 9 bp upstream of the 310 region, the second is separated from it by 5 bp and co-incides with the putative 335 region. These homologies suggested our study of the expression of the binase and RNase Bp genes in a set of B. subtilis strains with mutations in the regulatory protein genes of the PHO regulon. A BLAST sequence similarity search of the B. subtilis (strain 168) genome [13] using binase (X53697) and RNase Bp (U06867) sequences revealed that it contains no genes showing homology to the genes for these RNases. Expression of the barnase gene in B. amyloliquefaciens, its original source, is independent of inorganic phosphate and its promoter is quite di erent from that of binase and RNase Bp. In our experiments, the barnase gene was used as an additional control. Fig. 2 shows the binase and RNase Bp production Fig. 1. Promoters of binase and RNase Bp genes in comparison with phob and pst promoters of B. subtilis. In all promoters, 10 regions and mrna start sites are underlined, 6-bp sequences similar to consensus TTAACA are boldface printed. In binase and RNase Bp promoters, putative 35 regions are underlined. The binase promoter is a part of a sequence submitted to GenBank with accession number X53697, the RNase Bp promoter is a part of a sequence submitted to GenBank with accession number U06867, the phob promoter is from [5] and the pst promoter is from [14].

4 220 L.V. Znamenskaya et al. / FEMS Microbiology Letters 173 (1999) 217^222 Fig. 2. The e ect of inorganic phosphate on the binase and RNase Bp genes expression in B. subtilis strains. A: Growth (1) and biosynthesis of binase (2) by B. subtilis JH642 carrying pmz55. B: Growth (1) and biosynthesis of RNase Bp (2) by B. subtilis JH642 carrying pmz56. The time of P i addition is shown by the arrows. P i (100 mg/l) was added before the inoculation (squares) on the sixth (triangles) and 12th h of cultivation (white circles). Control (black circles) is without additional phosphate. from the plasmids pmz55 (with the whole binase gene) and pmz56 (with the whole Rnase Bp gene) in B. subtilis JH642, which is wild-type with respect to the PHO regulon. Addition of P i to the culture media in di erent growth phases resulted in the cessation of the biosynthesis of either ribonuclease. In order to determine if RNase Bp and binase expression is Pho-regulated, the phop mutant strain (MH5117) and the phor mutant strain (MH5124) were transformed by the plasmids pmz55, pmz56 and pmt420 (the whole barnase gene). As shown in Table 2, there was practically no RNase activity in B. subtilis phop and phor mutant strains from either the binase or Rnase Bp gene, while in the wild-type strain binase and RNase Bp activities were rather high. On the other hand, not inducible by phosphate limitation, barnase activity was high in wild-type and mutant strains both in presence and absence of inorganic phosphate. These data indicate that PhoP and PhoR proteins are required for binase and RNase Bp gene expression in B. subtilis. 4. Discussion Promoter regions of known B. subtilis PHO-regulated genes contain common elements necessary for the interaction with activated PhoP protein. Thus, the promoter of the phod gene contains a conserved 6-bp sequence located 9 bp 5P of a 310 consensus

5 L.V. Znamenskaya et al. / FEMS Microbiology Letters 173 (1999) 217^ Table 2 Expression of the genes for binase, RNase Bp and barnase in B. subtilis strains Strain Plasmid, gene OD 590 RNase, opt. u (ml h) 31 OD P i +P i 3P i +Pi JH642 pmz55 (binase) 4.0 þ þ þ þ 105 pmz56 (RNaseBp) 4.2 þ þ þ þ 237 pmt420 (barnase) 5.7 þ þ þ þ 321 MH5117 pmz þ þ þ þ 11 vpho P pmz þ þ þ þ 23 pmt þ þ þ þ 2220 MH5124 pmz þ þ þ þ 17 vpho R pmz þ þ þ þ 21 pmt þ þ þ þ 2117 JH642 ^ 6.30 þ þ þ þ 7.5 JH642 pub þ þ þ þ 12.2 JH642 pc þ þ þ þ 7.0 [12]. In the B. subtilis pst promoter, four 6-bp sequences separated by non-conserved 5 bp were found [14]. In the Pv promoter of the phob gene, there are three tandem TTAACA-like sequences separated by four or ve nucleotides. Liu and Hulett showed that the phosphorylated PhoP binds to the 318^373 region of the Pv promoter of the phob gene of B. subtilis and there are three tandem TTAACA-like sequences within the PhoP binding site [5]. The promoters of binase and RNase Bp also contain two of the TTAACA-like sequences in the same positions upstream of the 310 box as the rst two of the B. subtilis phob and pst promoters (Fig. 1). Although it seems clear that the PhoP protein is necessary for the control of these promoters, further experiments on direct binding of PhoPVP to promoters will demonstrate how many of the TTAACA-like repeats are involved in the interaction. The results presented here provide evidence that regulatory pathways homologous to those observed in the B. subtilis PHO regulon function to control ribonuclease production in some other bacteria of the genus Bacillus. Acknowledgments We are grateful to Dr F.M. Hulett for the B. subtilis JH642, MH5117 and MH5124 strains. We also thank Dr S. Kostrov and Dr T. Akimkina for helpful suggestions. References [1] Msadek, T., Kunst, F., Henner, D., Klier, A., Rapoport, G. and Dedonder., R. (1990) Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degs and degu. J. Bacteriol. 172, 824^834. [2] Hulett, F.M. (1996) The signal-transduction network for Pho regulation in Bacillus subtilis. Mol. Microbiol. 19, 933^939. [3] Hartley, R.W. (1997) Barnase and Barstar. In: Ribonucleases: Structures and Functions (D'Alessio, G. and Riordan, J.F., Eds.), pp. 51^100. Academic Press. [4] Znamenskaya, L.V., Gabdrakhmanova, L.A., Chernokalskaya, E.B., Leshchinskaya, I.B. and Hartley, R.W.(1995) Phosphate regulation of biosynthesis of extracellular RNases of endospore-forming bacteria. FEBS Lett. 375, 16^18. [5] Liu, W. and Hulett, F.M. (1997) Bacillus subtilis PhoP binds to the phob tandem promoter exclusively within the phosphate starvation-inducible promoter. J. Bacteriol. 179, 6302^ [6] Jones, D.H. and Howard, B.H. (1990) A rapid method for site-speci c mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles. Biotechniques 8, 178^183. [7] Znamenskaya, L.V., Gabdrakhmanova, L.A., Chernokalskaya, E.B. and Leshchinskaya, I.B. (1995) Regulation of the biosynthesis of extracellular ribonucleases in the native strains of Bacilli and recombinant strains of Escherichia coli. Microbiologiya 64, 616^622. [8] Je ris, G.D., Holtman, W.F. and Guse, D. (1957) Rapid

6 222 L.V. Znamenskaya et al. / FEMS Microbiology Letters 173 (1999) 217^222 method for determining the activity of microorganisms on nucleic acids. J. Bacteriol. 73, 61^79. [9] Glover, D.M. (1988) DNA cloning. A practical approach. IRL Press. [10] Sanger, F., Nicklen, S. and Coulson, A.R.. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463^5467. [11] An nsen, C.B., Red eld, R.R., Choate, W.I., Page, J. and Carrol, W.R. (1954) Studies of gross structure, cross-linkage and terminal sequences in ribonuclease. J. Biol. Chem. 207, 201^210. [12] Eder, S., Shi, L., Jensen, K., Yamane, K. and Hulett, F.M. (1996) A Bacillus subtilis secreted phosphodiesterase/alkaline phosphatase is the product of a Pho regulon gene, phod. Microbiology 142, 2041^2047. [13] Moszer, I., Glaser, P. and Danchin, A. (1995) SubtiList: a relational database for the Bacillus subtilis genome. Microbiology 141, 261^268. [14] Qi, Y., Kobayashi, Y. and Hulett, F.M. (1997) The pst operon of Bacillus subtilis has a phosphate-regulated promoter and is involved in phosphate transport but not in regulation of the pho regulon. J. Bacteriol. 179, 2534^2539.