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Genomics |
Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116, USA1
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany2
Unité de Biochimie Microbienne, D épartement des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, F-75724 Paris Cedex 15, France 3
Author for correspondence: Jörg Stülke. Tel: +49 9131 8528818. Fax: +49 9131 8528082. e-mail: jstuelke{at}biologie.uni-erlangen.de
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ABSTRACT |
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Keywords: sugar transport, PTS, phosphorylation, gene regulation, Bacillus subtilis
Abbreviations: EI and EII, Enzymes I and II; HPr, histidine-containing phosphoprotein; PTS, phosphotransferase system; PRD, PTS regulation domain
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Saier & Reizer, 1992 ).
In addition to its function in sugar transport, the PTS is one of the major signal transduction systems of the bacterial cell, as suggested by the multitude of regulatory functions exerted by PTS components. In both Gram-positive and Gram-negative bacteria proteins of the PTS are involved in carbon catabolite repression. They also control the induced expression of several catabolic operons in response to inducer availability by modifying the activities of transcriptional regulators, transport proteins and enzymes. Moreover, the PTS is involved in the regulation of nitrogen metabolism, chemotaxis towards carbohydrates and genetic competence (Postma et al., 1993 ; Powell et al., 1995 ; Saier & Reizer, 1994 ; Stülke & Hillen, 1998 ). Many, but not all of these regulatory processes involve phosphotransfer between PTS proteins and the proteins whose activities are modulated by the PTS. These phosphorylation reactions link the activities of the target proteins to PTS activity and thus to the availability of PTS substrates. Glycerol kinase from low-GC Gram-positive bacteria is phosphorylated by HPr in the absence of repressing carbon sources. This phosphorylation stimulates the catalytic activity of the enzyme (Charrier et al., 1997 ). Similarly, lactose transport activity in Streptococcus thermophilus is controlled by HPr-dependent phosphorylation of a IIA-like regulatory domain fused to the C-terminal end of the membrane-bound lactose:H+ symporter (Gunnewijk et al., 1999 ). Moreover, several positively acting transcriptional regulators have been shown to possess conserved PTS regulation domains (PRDs) (Tortosa et al., 1997 ; St ülke et al., 1998 ). The activator and antiterminator proteins that contain PRDs are often subject to dual control by the PTS: in the absence of the inducer of the controlled operon, they are phosphorylated and thus inactivated, but their activity may also depend on an additional HPr-dependent phosphorylation. This latter phosphorylation occurs only in the absence of a repressing carbon source, thus providing a means for hierarchical expression of genes required for the utilization of secondary carbon sources (for review see Stülke et al. , 1998 ).
The PTSs of enteric bacteria and of low-GC Gram-positive bacteria have been the subject of intensive investigation (see Saier & Reizer, 1994 ). More recently, the potential regulatory roles of PTS components of Mycoplasma spp., non-enteric Gram- negative bacteria such as Haemophilus influenzae and Alcaligenes eutrophus, and the high-GC Gram-positive bacterium Streptomyces coelicolor have been investigated (Zhu et al. , 1993 ; Macfayden et al., 1996 ; Pries et al., 1991 ; Titgemeyer et al., 1995 ). The advent of complete genome sequence analysis of the complete set of PTS proteins encoded within a given genome has resulted in the identification of several novel genes and proteins (see Reizer et al., 1996 ; Reizer & Reizer, 1996 ).
Here, we present an analysis of the complete set of genes encoding PTS and PTS-associated proteins in the model Gram-positive bacterium Bacillus subtilis. We show that B. subtilis encodes 15 complete PTS permeases of which only seven have been characterized previously. Sequence and functional analyses have allowed us to propose substrates for seven of the eight novel permeases. Moreover, 13 PTS- associated proteins are also encoded in the B. subtilis genome. The significance of these findings is discussed.
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METHODS |
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. Escherichia coli DH5
(Sambrook et al., 1989 ) and TGI (Gibson, 1984 ) were used for cloning experiments.
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gamP (ybfS) was disrupted as follows. A fragment of 977 bp internal to gamP was amplified by PCR using the oligonucleotides X1 (5'-GGGCGAGGGAATTCCGATTAT) and X2 (5'- CTGGTTCAGAAGCTTTGCCG). The PCR product was cut with EcoRI (generated by PCR) and HindIII and cloned into the integrative plasmid pHT181 (Lereclus & Arantè s, 1992 ) to give pX1. B. subtilis 168 was transformed with pX1 and the plasmid was integrated into the chromosome. The DNA of the B. subtilis strain harbouring pX1 was extracted, hydrolysed by EcoRI and religated. E. coli TGI was transformed with the ligation mixture, yielding plasmid pHT181::X1 carrying a 3 kb EcoRI fragment. This fragment contained the PCR-amplified region and about 2 kb upstream of that region. A StuI digestion of pHT181::X1 provided a fragment of 947 bp corresponding to the last 677 bp of gamP, the first 93 bp of gltP and 147 bp of the gamP/gltP intergenic region. This StuI fragment was then replaced by an aphA3 cassette (Trieu-Cuot & Courvalin, 1983 ) located on a 1·5 kb ClaI fragment, leading to plasmid pHT181::X1::aphA3.
ypqE was disrupted by the following approach. A fragment of 594 bp, carrying ypqE and 90 bp downstream of the stop codon of this gene was amplified by PCR using the primers ypq-1 (5'-GAAGAATTCATATGCTGAAAAAATTATTCGGAAT) and ypq-2 (5'- CCCCCCGGGTCGACTACAGT TTACCGAATATTTG). The amplified fragment was cloned into pUC19 at the EcoRI and SalI sites (sites were introduced with the PCR primers), to yield plasmid pXX1. A 1·5 kb ClaI fragment carrying aphA3 was inserted into pXX1 at the unique AvaI site, yielding plasmid pXX1::aphA3. The 2·1 kb EcoRI–SalI fragment carrying ypqE interrupted by aphA3 from pXX1::aphA3 was inserted into plasmid pHT181 between the EcoRI and SalI sites, giving pHT181::XX1::aphA3.
E. coli was grown in LB medium (Sambrook et al., 1989 ) whereas B. subtilis was grown in SP medium or C minimal medium (Martin-Verstraete et al., 1990 ). The minimal media were supplemented with auxotrophic requirements (at 50 mg l-1) and carbon sources as indicated. CSE is C medium supplemented with potassium succinate (6 g l-1) and potassium glutamate (8 g l-1). Doubling times were determined by growing the bacteria in C minimal medium supplemented with 0·2% (w/v) of the carbon source indicated. CSE containing 0·2% (w/v) glucitol was used for precultures. Agar plates were prepared by the addition of 17 g Bacto agar l-1 (Difco) to LB, SP or C medium.
DNA manipulation, transformation and phenotypic characterization.
DNA extraction and manipulation were performed using standard procedures (Sambrook et al., 1989 ). Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturers.
Standard procedures were used to transform E. coli and transformants were selected on LB plates containing ampicillin (100 µg ml-1) or spectinomycin (100 µg ml-1) (Sambrook et al., 1989 ). B. subtilis was transformed with chromosomal or plasmid DNA according to the two-step protocol described by Kunst & Rapoport (1995) . Transformants were selected on SP plates containing spectinomycin (100 µg ml -1), chloramphenicol (5 µg ml-1 ) or kanamycin (5 µg ml-1).
Computer-aided analyses.
Database searches were performed using the BLAST 2.0 program (Altschul et al., 1990 ) on the NCBI server (http://www.ncbi.nlm.nih.gov/blast/). The organization of B. subtilis gene clusters was determined using the SubtiList database (Moszer et al., 1995 ).
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RESULTS AND DISCUSSION |
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and 3 list all recognized PTS proteins and PTS- associated proteins encoded within the B. subtilis genome. Interestingly, only one complete EI-like protein and just two HPr-like proteins, i.e. HPr and Crh, are present (see Tables 2 and 3). By contrast, the E. coli chromosome encodes five EI paralogues (EI, EIAni, EINtr and the two EI domains in the multidomain proteins ADI and TTP) and five paralogues of HPr (HPr, FPr, NPr, DPr, TPr) (Blattner et al., 1997 ; Reizer & Saier, 1997 ). Domains similar to the phosphohistidine domain of phosphoenolpyruvate synthase (similar to EI) are attached to the C termini of pyruvate kinases of several Gram-positive bacteria, including B. subtilis (Table 3), Bacillus stearothermophilus and Lactobacillus delbrueckii (Sakai & Ohta, 1993 ; Nguyen & Saier, 1995 ; Branny et al., 1996 ). The presence of multiple paralogues of EI and HPr in E. coli and the scarcity of these paralogues in B. subtilis suggests that the latter organism evolved alternative mechanisms to satisfy the specific regulatory and/or functional roles of these proteins.
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encodes proteins that fall into the Glc family. Two of these permeases (PtsG and GamP) exhibit the IICBA domain structure, whereas the remaining two permeases (MalP and NagP) lack a IIA domain and exhibit the IICB domain order. Two genes (yyzE and ypqE) encode two distinct IIA Glc-like proteins. YpqE is a full length paralogue (168 residues) that presumably functions with the IICB permease(s) of this family or the IIBC permease(s) of the Scr family. YyzE, the second IIAGlc -like protein, is a truncated IIA homologue (76 residues) possessing the two active site histidyl residues but lacking about 70 and 30 residues corresponding, respectively, to the N and C termini of the orthologous crr gene product of enteric bacteria (Nelson et al., 1984 ). It is not known if YyzE is functional in the PTS phosphorylation cascade. nagP, encoding a putative permease for N-acetylglucosamine, forms a monocistronic transcriptional unit and is not linked to other genes involved in the utilization of N-acetylglucosamine (see Fig. 1a). malP is the distal gene of a presumptive three-gene operon encoding also a phospho-- glucosidase (MalA) that has the capacity to hydrolyse phosphorylated maltose generated by MalP (Fig. 1b). gamP required for the utilization of glucosamine (see below) is the second gene of a two-gene operon. The proximal gene of this operon, gamA (ybfT ), encodes a putative glucosamine-6-phosphate-deaminating isomerase. Divergently transcribed relative to the putative gamAP operon is a gene encoding a transcriptional repressor of the GntR family (see Fig. 1c). ypqE seems to form a monocistronic unit (Fig. 1d) while yyzE is followed by bglA encoding a phospho- ß-glucosidase (Fig. 1e) (Zhang & Aronson, 1994 ).
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, section 3). Four of these (SacP, SacX, TreP and YbbF) have the IIBC domain structure. The fifth member of this family, BglP, has the IIBCA domain order. Altogether, the B. subtilis chromosome possesses nine genes encoding nine pairs of IIB and IIC domains that belong to the glucose and sucrose families. By contrast, only five IIAGlc -like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the IIAGlc-like proteins or domains must function in transfer of the phosphoryl group to more than one IIBGlc- and/or IIBScr-like proteins as was previously suggested with the IIAGlc domain of the glucose-specific EII complex (Sutrina et al., 1990 ). The operons containing the genes encoding EIIs for sucrose, trehalose and aryl-ß-glucosides have all been described (Fouet et al., 1987 ; Zukowski et al., 1990 ; Schöck & Dahl, 1996 ; Le Coq et al., 1995 ). ybbF, encoding a novel EII of the Scr family, is the fifth gene in a putative eight-gene operon (Fig. 2). Based on the fact that one of the genes (ybbD) of this gene cluster encodes a ß-glycosidase homologue, we propose that YbbF is involved in the utilization of unknown ß-glucosides.
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, section 4) includes two EII complexes each encoded by three distinct genes (licBCA and ydhMNO). Thus, YdhMNO and LicBAC are homologous to the IIB, IIA and IIC proteins that constitute the lactose-specific PTS permeases of Gram-positive bacteria (Lengeler et al., 1994 ). The Lic-PTS is encoded within the licBCAH operon that also includes a phospho- ß-glucosidase encoding gene, licH (Tobisch et al. , 1997 ). The ydhMNO genes are proximal in a putative eight-gene operon that also encodes a ß-1,4-mannosidase, a ß-glycosidase, a mannose-6-phosphate isomerase and a fructokinase (Fig. 3a). Based on these observations we propose that the Ydh-PTS is involved in the transport of oligo-ß- mannosides. This would be analogous to the Lic-PTS which transports oligomeric ß-glucosides. Indeed, the ydhMNO operon was recently shown to be involved in mannan utilization and to be induced by konjac glucomannan (Sadaie & Yata, 1998 ). A paralogous IIC domain (YwbA), the function of which is yet to be determined, is an additional member of the B. subtilis lactose permease family. ywbA is linked to a gene of unknown function and it is not clustered to any other gene involved in carbohydrate utilization (Fig. 3b).
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, section 5) is a fructose-6- phosphate-forming fructose permease of the Man family. It is the only member of the Man family present in B. subtilis (Martin- Verstraete et al., 1990 ).
The Fru family (Table 2, section 6) includes the following three permeases, each containing three domains (IIA, IIB and IIC) that reside on a single polypeptide chain: (i) MtlA with a CBA domain order homologous to the E. coli mannitol permease; (ii) FruA with an ABC domain sequence homologous to the fructose-1-phosphate-forming fructose-specific PTS permease of mycoplasmas (Reizer et al., 1996 ); and (iii) ManP (YjdD) with a BCA domain order. All three domains of FruA and the putative mannose permease (ManP) exhibit significant sequence similarity with protein domains of the fructose- specific permease (IIABCFru; fructose-1-phosphate-forming) of mycoplasmas (Reizer et al., 1996 ). mtlA , encoding EIIMtl, is the first gene in a bicistronic operon which also encodes mannitol-1-phosphate dehydrogenase (Fig. 4a). manP is the first gene in a putative tricistronic operon. The second gene encodes a mannose-6- phosphate isomerase, whereas the function of the third gene is unknown (Fig. 4b). fruA, encoding the fructose permease, is the distal gene in a tricistronic operon. The first gene of this putative operon, fruR, encodes a repressor of the DeoR family and the second gene, fruB, encodes an orthologue of the E. coli fructose-1-phosphate kinase (Fig. 4c).
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; Nobelmann & Lengeler, 1995 ) are not present in B. subtilis. Furthermore, none of the sugar-specific EII complexes of B. subtilis exhibit a mosaic structure like that observed for the Sgc permease of E. coli (Reizer et al., 1996 ). We also note that PTS permeases of the Scr family have the BC(A) domain order, members of the Glc family exhibit the CB(A) domain order and members of the Fru family have variable domain orders (IICBA, IIBCA and IIABC). Finally, the B. subtilis EII complexes of the Lac and Man families consist of single domain proteins.
PTS-associated proteins
Expression of several catabolic operons in B. subtilis is controlled by PTS-dependent phosphorylation of inducer-generating enzymes or transcriptional regulators (Deutscher et al., 1997 ; Stülke et al., 1998 ).
Glycerol utilization in B. subtilis depends on a functional PTS, although glycerol is transported into cells by a non-PTS permease (Reizer et al., 1984 ; Beijer & Rutberg, 1992 ). Mutations that bypass this dependency were isolated and found to be in glpK, the gene encoding glycerol kinase which generates glycerol 3-phosphate, the internal inducer of the glp regulon in B. subtilis (Wehtje et al., 1995 ). Subsequently, direct phosphorylation of glycerol kinase by EI and HPr was demonstrated (Charrier et al., 1997 ).
A protein domain, PRD, is found in transcriptional regulators such as RNA-binding antiterminators and DNA-binding activators that serves as a target of PTS-dependent phosphorylation (Tortosa et al., 1997 ; Reizer & Saier, 1997 ; Stülke et al., 1998 ). PRD- containing regulators are found primarily in Gram-positive bacteria. Eight PRD-containing regulators are encoded within the B. subtilis chromosome (see Table 3). All eight proteins regulate the expression of genes encoding PTS permeases and associated catabolic enzymes. PRD-containing transcriptional antiterminators and the activator protein LevR have been studied extensively (Deutscher et al., 1997 ; Martin-Verstraete et al., 1998 ; Stülke et al., 1998 ). Four antiterminators of this type are present in B. subtilis (see Table 3). They control the expression of genes required for glucose, sucrose and ß-glucoside utilization (St ülke et al., 1997 ; Steinmetz et al., 1989 ; Schnetz et al., 1996 ). In addition, PRD-containing transcriptional activators, LevR and LicR, control expression of the levanase and licBCAH operons, respectively (Débarbouill é et al., 1991 ; Tobisch et al., 1997 ). The LevR activator contains two PRDs. LicR contains two PRDs and a C-terminal domain homologous to EIIAFru. The phosphorylation site in this domain is conserved and a mutation of this site was shown to result in constitutive LicR activity (Tobisch et al., 1999 ). The complete nucleotide sequence of the B. subtilis genome revealed the presence of two novel putative transcriptional activators that are similar to LicR. Since yjdC is located just upstream of the operon involved in mannose utilization (see Fig. 4b), the designation ManR was proposed (Stülke et al., 1998 ). YdaA is homologous to the regulators of mannitol catabolism of B. stearothermophilus (Henstra et al., 1999 ) and Clostridium acetobutylicum (accession no. U53868). The designation MtlR was thus proposed for YdaA (Stülke et al. , 1998 ).
Phenotypes of B. subtilis strains defective in EII components
The functional role of the GlvC and YbfS permeases was determined. glvC was identified in the framework of the B. subtilis sequencing project and proposed to encode a ß-glucoside- specific EII (Yamamoto et al., 1996 ). However, the functional characterization of genes encoding a maltose-specific EII and a phospho--glucosidase from Fusobacterium mortiferum revealed that the homologous GlvA of B. subtilis, encoded in the same operon as GlvC, is in fact a phospho--glucosidase rather than a phospho-ß-glucosidase (Bouma et al., 1997 ; Thompson et al., 1998 ). In addition, GlvC exhibits higher sequence similarity to PTS permeases of the glucose/maltose family than to the sucrose/ß-glucoside-specific PTS permeases. To study the functional role of glvC we constructed a glvC mutant of B. subtilis. We additionally constructed strains containing ypqE and ybfS mutations (Kunst et al., 1997 ; see Table 2). To obtain a comprehensive set of mutants we used strain GP113 in which the part of ptsG encoding the IIB and IIA domains of the glucose permease was deleted. The IIA domain of the glucose permease has been shown to phosphorylate IIB domains of the sucrose- and trehalose-specific PTS permeases that lack a IIA domain (Sutrina et al., 1990 ; Dahl, 1997 ).
B. subtilis 168 and the mutant strains were grown in C minimal medium with either glucose, maltose, glucosamine or N- acetylglucosamine as a single source of carbon and energy and their doubling times were determined (Table 4 ). All four sugars were readily utilized by the wild-type strain, although the generation time of cultures grown on N-acetylglucosamine was longer than the generation time of cells grown on either glucose, maltose or glucosamine. The PTS is required for efficient catabolism of all the carbon sources tested as is evident from the slow growth rate (or the lack of growth) of the ptsH mutant strain. As observed previously, the ptsH mutant strain grew slowly on glucose, suggesting the presence of a non-PTS system for glucose transport and phosphorylation (Stülke et al., 1995 ). The recently identified glucose transporter, GlcP, is presumably responsible for this residual growth (Paulsen et al., 1998 ). Similarly, slow growth of the ptsH mutant was observed with glucosamine, whereas maltose and N-acetylglucosamine were not utilized by this strain. The IIA and IIB domains of the glucose permease were required for efficient growth on glucose and maltose while the ptsG mutation did not affect growth on glucosamine or N- acetylglucosamine. Four conclusions can be drawn from these results. (i) PTS permeases distinct from PtsG may transport and phosphorylate glucose, albeit with low efficiency. (ii) Phosphotransfer via the IIA Glc domain appears to be necessary for efficient utilization of maltose. (iii) The glvC mutant strain GP110 grew normally on all the carbon sources tested with the exception of maltose. We propose, therefore, that glvC encodes a maltose-specific PTS permease and we redesignate the gene malP. This finding is in agreement with the presence of a phospho--glucosidase-encoding gene within the malP operon (see Fig. 1b) and with the strong sequence conservation of MalP with other maltose- and glucose-specific PTS permeases. (iv) The ybfS mutation resulted in impaired growth in the presence of glucosamine, suggesting that the EII encoded by ybfS catalyses the transport and phosphorylation of this sugar. This observation is reinforced by the genetic arrangement. ybfT, which precedes ybfS, exhibits a high degree of sequence similarity to the characterized (deaminating) glucosamine-6- phosphate isomerase of E. coli (Fig. 1c). Therefore, we propose to designate the ybfT and ybfS genes as gamA and gamP, respectively. The ypqE mutation did not yield a recognizable phenotype under the conditions tested here and the function of the encoded protein therefore remains unknown.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 July 1999;
revised 31 August 1999;
accepted 3 September 1999.
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-independent terminator is located downstream of malP . (c) Genetic map of the gam gene cluster. A potential 
