MICROBIOLOGY LETTERS

FEMS Microbiology Letters 132 (1995) 79-83

Contributions of XylR, CcpA and HPr to catabolite repression of the xyl operon in Bacillus subtilis

Michael K. Dahl, Wolfgang Hillen *

Lehrstuhlfir Mikrobiologie, Institutftir Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander Universitiit Erlangen-Niirnberg. Staudtstr. 5, 91058 Erlangen, Germany

Received 14 June 1995; accepted 1 August 1995

Abstract

The xyl operon of Bacillus subtilis is regulated at the level of transcription by xylose induction via the Xyl repressor and by catabolite repression. We have investigated the influence of ccpA, ptsH, ptsG and .xylR mutations on catabolite repression of xyL4 expression. The results indicate that full glucose repression of the xyf operon requires CcpA, Hpr and XylR. In contrast, fructose repression depends on CcpA and Hpr, but not on XylR. The ptsHl mutation relieves catabolite repression only partially, suggesting the possibility that other presently unknown signals are sensed by CcpA.

Keywords: Xylose utilization; Gene regulation; Repression of transcription; Repressor-operator interaction; BacilLs subtilis

1. Introduction

Xylose utilization in Bacillus subtilis is encoded by an operon containing at least two genes, .@A (encoding xylose isomerase) and xylB (encoding xylulose kinase) .(see Fig. 1) [l-3]. The ope.ron is negatively regulated at the level of transcription by the Xyl repressor (XylR) [4]. In the absence of the inducer xylose, the repressor turns down transcrip- tion of the xyl operon by interaction with a tandem xyl operator structure composed of two overlapping XylR binding sites, 0, and 0, [5]. Similar xylose utilization systems have been described for Bacillus megaterium [6] and Bacillus lichenifonnis [7]. The Xyl repressor proteins exhibit considerable sequence

?? Corresponding author. Tel: +49 (9131) 858081; Fax: +49 (9131) 858082; E-mail: hillen@imbg.bio1ogie.unierlangen.de

similarity in the range of 70% amino acid identity. They are also similar to glucokinase (GlcK) from Streptomyces coelicolor [f&9].

Regulation of the xyf operon is catabolite re- pressed depending on the presence of a cis acting catabolite responsive element (CRE) located in the coding sequence of xylA [ 10,111 and the trans acting protein CcpA [12]. CcpA interacts with HPr, the central component of the phosphoenolpyruvate phos- photransferase system [13], when it is phosphory- lated at Ser46 [13-151. The participation of XylR in glucose mediated repression was demonstrated in vitro, showing that glucose is an anti-inducer for the B. licheniformis encoded Xyl repressor [16] and in vivo in B. subtifis Ill].

We determine here the contributions of ccpA, ptsH and xylR to catabolite represssion of xylA exerted by glucose and fructose.

0378-1097/95/$09.50 0 1995 Federation of European Microbiological Societies. All rights reserved SSDI 0378-1097(95)00291-X

80 M. K. Dahl, W. Hillen / FEMS Microbiology Letters 132 (I 995) 79-83

0 EIIAG’C

xy/A

CRE

($-=J!& xylose

Fig. I. Genetic organization of the xyl operon from B. subrilis composed of xylA and xylB and the divergently oriented gene

xylR (encoding Xyl repressor). Under uninduced conditions, the Xyl repressor (R) exhibits a conformation (circle) able to bind the

xyl operators 0,. and O,,. Xylose interacts with XylR to induce

the expression of the xyl operon. Xylose bound XyIR (square) is

not able to interact with xylU. A catabolite responsive element

(CRE) is located within the coding sequence of .@A [I I]. Serine

46 (S46) phosphorylated HPr interacts with CcpA leading to

catabolite repression [ 131. The phosphorylation of HPr at S46 is

catalyzed by a protein kinase (K) in the presence of ATP, dephosphorylation is catalyzed by a phosphatase (Pase) [I 31. HPr

is also phosphorylated at histidine 15 (H15) by the P-EI. This

phosphate is transferred to enzymeflAt”‘.

Table I Bacillus subtilis strains used in this study

2. Materials and methods

2.1. Materials and general methods

Materials, enzymes and media used in this study have been described earlier [4,5]. If necessary we used chloramphenicol (5 pg/ml), erythromycin (25 pg/ml), kanamycin (5 pg/ml) or spectinomycin (100 pug/ml) for selection in B. subtilis, and ampi- cillin (100 pg/ml) for selection in E. coli.

2.2. Bacterial strains and plasmids

All bacterial strains used and constructed in this study are listed in Table 1. E. coli strain DHSa [ 171 was used as a general cloning host. Plasmid prepara- tions were done as described [ 171. B. subtilis strain WH366 was constructed by integration of plasmid pWH760 (see below) into the amyE gene of B. subtilis wild-type followed by kanamycin selection and screen for amyE minus phenotype on LB 0.2% starch plates using Lug01 reagent (Merck, Darmstadt, FRG). Competent B. subtilis cells were obtained by

Strain Marker Source or reference a

B. subtilis I68 GM1222 MD156

MZ303

QB6046 WH340

WH366 WH369

WH370

WH37l

WH372 WH373

WH374 WH375 WH376

trpC2 trpC2 pheA1 A(bgaX) amyE::(gntRK-lucZ) ptsHI ’ (Cm’)

trpC2 ccpA::Tn9/7A(erm-lacZ)::spe p?sH::cat ’ sacT30ptsG::cat amyE::(sucP’-lacZ aphA3) B. subtilis WH335 xylR mutant xylR::Erm’

amyE::( Pxyl,,,,, EcoRI-HindIII-spoVG-IacZ Kn’) trpC2 WH366 xylR::erm WH366 ptsG::cat WH366 pisH::cat ’ WH366 ccpA::Tn917A(erm-1ucZ)::spe WH366 pisH1 ’ (Cm’) WH366 xylR::ermptsH::cat ’ WH366 ccpA::Tn 917A(erm-1acZ):: spe xylR::erm WH366 xylR::erm ptsH1 ’ (Cm’)

BGSCIAlh

II41

1221 I231 1231 1101 This study ’ WH340 tf --) WH366

QB6046 tf -+ WH366 MZ303 tf --) WH366

MDIS6tfdWH366 GM I222 tf + WH366 MZ303 tf -+ WH369

MD156 tf -+ WH369 WH340 tf -f WH373

a tf + indicates transformation of chromosomai DNA. h BGSC, Bacillus Genetic Stock Centre. Columbus, Ohio.

mutation ptsH1 leads to an amino acid exchange S46A [I41 ” pfsH::cat corresponds to the deletion of a I55-bp HindIII-HpaI fragment within the ptsH gene which was replaced by the cat gene of pC 194. This construct allows expression of ptsl [23]. ’ for detailed description of construction see Materials and methods.

M. K. Dahl, W. Hillen / FEMS Microbiology Letters 132 (1995) 79-83 81

a one-step procedure as described [18] and used to transform chromosomal or plasmid DNA, respec- tively, for strain construction.

Construction of plasmid pWH760 was carried out in the following way: a 2288 bp EcoRI-Sac1 frag- ment from pWH484 [l 11 was ligated to the 8642 bp EcoRI-Sac1 fragment of pMD429 [19].

2.3. /3-galactosiahse assays

/3-Galactosidase activity was determined using B. subtilis strains as indicated. The cells were harvested at an OD,, of 0.5, and the fl-galactosidase activities were monitored according the method of Miller [20] with modifications as described previously [21]. Cells were grown in C minimal medium [21] containing tryptophan (50 pug/ml), ferric citrate (11 pg/ml), magnesium chloride (2 mM), calcium chloride (1 mM) and 0.4% K-glutamate. To study the induction by xylose and repression by glucose or fructose, the medium was supplemented with sugars in concentra- tions and combinations as indicated.

3. Results and discussion

We have quantitated in vivo the roles played by several genes contributing to catabolite repression.

For that purpose we have constructed strains carry- ing xylR, ccpA, ptsG and ptsH mutations in single copy &A-1acZ background as shown in Table 1. The xylA-lacZ transcriptional fusion contains CRE and xyl0 leading to xylose induction and glucose or fructose repression as shown in Table 2. Induction by xylose and repression by glucose or fructose was studied in the presence of 10 mM of the respective sugars. The results are depicted in Table 2. Cells harboring the xylA-1acZ fusion in a wild-type back- ground exhibit a 58-fold induction by xylose [ 11,161. Strains lacking a functional enzymeIIG’c ( AptsG) show no glucose repression since glucose cannot be taken up. Repression by fructose is unaffected in this strain. A AptsH strain (WH371) is unable to take up glucose or fructose and, hence, no repression by fructose was detectable, whereas slight glucose re- pression is still observed, which is probably medi- ated by XylR. The involvement of XylR in glucose repression has been demonstrated in vitro and in vivo [ 11,161. Deletion of xylR leads to constitutive expression of the xylA-1acZ fusion and to a reduced efficiency of glucose repression, whereas repression by fructose is not affected (see Table 2). We assume that fructose and glucose can trigger catabolite re- pression and that glucose, in addition, effects induc- tion of xylA via interaction with XylR [11,16]. Catabolite repression depends on CcpA. This conclu-

Table 2 fi-Galactosidase activities and factors of regulation of a @A:: 1acZ fusion

Strain/relevant genotype Uninduced *

WH366 (wild-type) 11.4rt2.1 WH369 AxylR 842 f 100 WH370 AptsC 16.2 f 7.4 WH371 AptsH 22.2 f 1.0 WH372 AccpA 22.7 f 5.0 WH373 ptsH1 21.3 f 1.0 WH374 AxylR AptsH 119Ok68 WH375 AxylR AccpA 641 f 12 WH376 AxylR ptsHl 1250 f 146

Induction b by xylose

626 f 10 (58 f 12) 915 f 36 (1.1 + 0.1) 509 f 24 (37 f 12) 1062 f 60 (48 &- 2.2) 809 f 65 (38 f 10) 706 f 40 (35 f 2.3) 1077 f 140 (0.9 f 0.1) 620*64(1.OItO.l) 886 f 101 (0.7 f 0.1)

Repression ’ by glucose

34.6 f 1.5 (18.1 f 0.6) 3 15 f 20 (2.9 L- 0.3) 596+34(0.8+_0.1) 713 f 88 (1.5 f 0.1) 251 f 19 (3.2 f 0.2) 84 f 3 (8.4 f 0.2) 1455 f 200 (0.7 + 0. I ) 484 f 53 (1.3 + 0.3) 281 + 53 (3.2 f 0.9)

Repression * by fructose

295 f 41 (2.2 f 0.9) 436k23c2.1 fO.l) 247 f 13 (2.1 + 0.1) 1034*34(1.0f0.I) 670~20(1.2~0.1) 563 f 27 (1.2 f 0.1) 1299 f 104 (0.8 f 0.2) 1277*162(0.5fO.l) 745 f 73 (1.2 f 0.2)

’ P-Galactosidase activities in minimal medium C with supplements as described in Materials and methods without xylose, glucose or fructose. ’ /3-Galactosidase activities in minimal medium C containing 10 mM xylose; factors of induction am presented in parenthesis ( P-galactosi- dase activity in the presence of xylose divided by activity in the absence of xylose.). ’ P-Galactosidase activities in minimal medium C containing 10 mM xylose and 10 mM glucose; factors of re.pmssion ate presented in parenthesis ( P-galactosidase activity in the presence of xylose divided by activity in the presence of xylose and glucose). d P-Galactosidase activities in minimal medium C containing 10 mM xylose and 10 mM fructose; factors of repression am presented in parenthesis ( /3-galactosidase activity in the presence of xylose divided by activity in the presence of xylose and fructose).

82 M.K. Dahl. W. Hillen / FEMS Microbiology Letters 132 (1995) 79-83

sion is supported by results obtained with a combina- tion of deletions in xylR and ccpA showing repres- sion by neither glucose nor fructose (Table 2). A ccpA deletion alone relieves fructose repression, but a residual 3.2-fold glucose repression is still de- tectable. This result can again be explained by XylR mediated glucose repression [ 11,161.

An exchange of serine 46 to alanine (ptsH1, see Table I> had no effect on the uptake of the F’TS-sub- strates glucose and mannitol or the non-F’TS sub- strates gluconate and glucitol [14] but the mutation abolished catabolite repression exerted by glucose on gluconate kinase, glucitol dehydrogenase and the mannitol specific catabolic enzymes [ 141. Glucose repression of xylA-lad expression is only partially relieved in a ptsHl strain (Table 2) as was observed for the inositol dehydrogenase 1141, whereas repres- sion by fructose is completely abolished. Since the ptsHl strain is able to take up glucose this result is in agreement with a XylR dependent glucose effect. Residual glucose repression is still observed in a ptsHI strain with a xylR deletion. Since glucose repression is completely relieved in a strain lacking CcpA and XylR these results suggest additional fac- tor(s) mediating catabolite repression via CcpA. Such facto&) could be intermediates of glycolysis and/or an additional protein. It has been postulated that CcpA may be able to sense the status of glycolysis by interaction with seryl46-phosphorylated HPr [ 131. On the basis of the results presented here we suggest the possibility that CcpA may be able to sense other signals as well.

Acknowledgements

This work was supported by grants from the EC (BI02-CT92-01371, the Deutsche Forschungsge- meinschaft and the Fonds der chemischen Industrie.

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