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1 Laboratorio de Microbiología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (1428), Argentina
2 Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain
Correspondence
Carmen Sánchez-Rivas
sanchez{at}qb.fcen.uba.ar
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The GenBank accession number for the sequences reported in this paper is AY211495.
Present address: Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This species is a low-G+C Gram-positive bacterium that has been divided into five DNA homology groups (Krych et al., 1980; Rippere et al., 1997). One of them, group II, has been separated in two subgroups, IIA and IIB (Krych et al., 1980). Many biotechnologically interesting strains have been included in the IIA subgroup, which can synthesize insecticidal proteins that are active against mosquito larvae, in particular against various species of the genera Culex and Anopheles. These species are known to be regular transmission vectors of filariasis and malaria (Baumann et al., 1991; Porter et al., 1993; Charles et al., 1996).
Although these bacteria can metabolize a wide variety of organic compounds and amino acids, they are unable to use hexoses and pentoses as sole carbon source (Russell et al., 1989; Alexander & Priest, 1990). For this reason, proteinaceous media, which are more expensive than alternative media based on starch or molasses, are used for toxin production (Russell et al., 1989; Couch, 2000). Consequently, studies disclosing the true metabolic potential of B. sphaericus are essential for genetic and industrial improvement programmes.
The inability to metabolize carbohydrates in this species has been attributed to the absence of key enzyme activities in both the Embden–Meyerhof–Parnas [phosphoglucoisomerase (EC 5.3.1.9), 6-phosphofructokinase (PFK) (EC 2.7.1.11)] and the Entner–Doudoroff [phosphogluconate dehydratase (EC 4.2.1.12) and phospho-2-keto-3-deoxygluconate aldolase (EC 4.1.2.14)] pathways (Russell et al., 1989). It has also been described that B. sphaericus was unable to transport either glucose or sucrose (Russell et al., 1989). However, we have recently shown PFK activity and we have sequenced the pfk gene present in all the homology groups of this species (Alice et al., 2002). Nevertheless, micro-organisms can hardly utilize sugars without a proficient sugar transport system. In bacteria, the phosphoenolpyruvate (PEP) : sugar phosphotransferase system (PTS) is a widespread and efficient means of translocating carbohydrates into the cytoplasm (Romano & Saier, 1992). The PTS consists of two general cytoplasmic proteins, the enzyme I (EI) and the heat-stable protein (HPr), which in turn transfers a phosphate group from PEP to the sugar specific components of the PTS, EIIsugar. This last element consists of three or four different proteins, often fused and found as domains of larger proteins (Postma et al., 1993). Then, the sugar-specific elements mediate sugar transport and its concomitant phosphorylation. PTS elements are also involved in other important functions, such as the general regulatory process of carbon catabolite repression (CCR) (Saier et al., 1996; Stülke & Hillen, 1999; Titgemeyer & Hillen, 2002).
In low-G+C Gram-positive bacteria, HPr can be phosphorylated in two residues: His15 and Ser46. PEP-dependent phosphorylation by EI occurs at the His15 site and this phosphoryl group is transferred to the EIIsugar (Postma et al., 1993). P-His15-HPr can also transfer its phosphoryl group to other non-PTS proteins, such as enzymes like glycerol kinase (Charrier et al., 1997) or regulators. These are antiterminators and transcriptional activators that possess PTS regulatory domains (PRD) which contain histidine residues phosphorylatable by P-His15-HPr and also by other components of the PTS (Stülke et al., 1998).
ATP-PPi dependent phosphorylation of HPr at Ser46 is catalysed by the crucial enzyme HPr-kinase/phosphatase (HPrK/P) (Galinier et al., 1998; Reizer et al., 1998; Kravanja et al., 1999; Mijakovic et al., 2002). HPrK/P senses the physiological state of the cell and changes its activity based on internal concentrations of ATP and glycolytic intermediates, such as fructose 1,6-bisphosphate (FBP). In conditions of high energetic metabolism, the kinase activity is stimulated and P-Ser46-HPr would be predominant, inhibiting phosphorylation at His15 and affecting all the events depending on P-His15-HPr. In low metabolic conditions, the drop in ATP and rise of phosphate concentration was found to stimulate the HPr phosphatase activity (Jault et al., 2000; Dossonnet et al., 2000; Mijakovic et al., 2002). However, HPrK/P from Mycoplasma pneumoniae exhibits an inverse mode of regulation, since this protein is by default a kinase rather than a phosphatase (Steinhauer et al., 2002). P-Ser46-HPr acts as a co-factor of the catabolite control protein A (CcpA), which forms a DNA-binding complex that recognizes specific sequences called cre sites (catabolic repression element) present in the promoter regions or structural part of genes subject to catabolite repression or activation (Henkin et al., 1991; Deutscher et al., 1995; Fujita et al., 1995; Titgemeyer & Hillen, 2002).
Since B. sphaericus does not use either hexoses or pentoses as a carbon source but possesses an enzyme activity used in glycolytic pathways (Alice et al., 2002), we decided to investigate whether a PTS was present in this species. The presence of general elements (HPr and EI) of the PTS would be evidence that a sugar transport system and/or a catabolite regulation pathway might be present in this micro-organism.
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. B. sphaericus strains were grown at 32 °C in Luria–Bertani (LB) medium or CTB minimal medium with aeration. CTB minimal medium was C medium (Martin-Verstraete et al., 1990) supplemented with thiamin (0·02 %), biotin (0·02 µg ml-1) and the carbon source as indicated in the text. Bacillus subtilis, Escherichia coli and Staphylococcus aureus were grown at 37 °C in LB medium. When appropriate, ampicillin (100 µg ml-1), kanamycin (5 µg ml-1), X-Gal (40 µg ml-1) and IPTG (1 mM) were added.
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). The fragment obtained was cloned into pGEM-T to create pALE-3. A cassette encoding kanamycin resistance was obtained from plasmid pDG783 (Guérout-Fleury et al., 1995), digested with EcoRI and ligated to pALE-3 digested with the same enzyme. The plasmid constructed (pALE-66) was used to transform B. sphaericus strains 2362 and ASB13052.
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. The samples were then immediately diluted with 2 ml LB and incubated at 32 °C for 90 min. Chromosomal transformants that had arisen from a double recombination through each of the ptsH arms flanking the kan gene were selected on LB agar plates containing kanamycin. However, the restriction-deficient strain only yields kanamycin-resistant colonies. After PCR and Southern blot analysis, one clone containing the right construction (ALE-7) was selected and further studied.
Cloning, DNA sequencing and sequence analysis.
Total DNA was isolated from B. sphaericus as described before (Alice et al., 2002) and used as template in standard PCR reactions with the primers described (Table 2, Fig. 1
). Amplified DNA fragments were cloned in pGEM-T and sequenced. Isolation of plasmid DNA from E. coli, enzymic digestions, ligations and inverse PCR reactions were performed by standard procedures (Sambrook et al., 1989). DNA sequencing was carried out by using an ABI PRISM dRhodamine Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase and an automatic ABI 310 DNA sequencer (Applied Biosystems).
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). Multiple sequence analysis was done using CLUSTAL X (Thompson et al., 1997).
Purification of HPr(His6) from B. sphaericus 2362.
The B. sphaericus 2362 ptsH gene was amplified by PCR with primers PtsHN and PtsHCH, containing BamHI and HindIII restriction sites respectively (Table 2, Fig. 1). This fragment and plasmid pQE30 (Qiagen) were digested with BamHI and HindIII, ligated with T4 DNA ligase and used to transform E. coli DH5
. A clone with the right construction (plasmid pALE-12) was grown in LB medium with ampicillin to OD600 0·6 and expression of the insert induced by adding 1 mM IPTG. The incubation was continued for 4 h and the protein purified with Ni-NTA Spin Columns (Qiagen) as described by the manufacturer. The purity of the protein was verified by SDS-PAGE, where B. sphaericus HPr(His6) represented >95 % of total protein (data not shown).
Site-directed mutagenesis of HPr and purification of HPr(His6)S46Q protein.
The site-directed mutagenesis to obtain the HPr mutant with the phosphorylation site Ser46 replaced by Gln was performed as described by Landt et al. (1990). Briefly, a PCR reaction was performed with chromosomal DNA from 2362 as template and primers PtsHN and PtsS46Q. The fragment obtained was purified and utilized as forward primer in a second PCR with the same template and PtsHCH as reverse primer. The new fragment amplified was cloned into pGEM-T to obtain plasmid pALE-2. It was digested with BamHI and HindIII and the mutated ptsH gene was ligated with pQE30 (Qiagen) digested with the same enzymes. One clone with the right construction, plasmid pALE-8, was selected and used to express and purify the HPr(His6)S46Q protein as described above for the wild-type protein.
Complementation assays.
In order to measure PTS activity, in vitro complementation assays were performed with cell extracts from S. aureus mutant strains. Strain S797A lacks a functional HPr and strain S710A lacks a functional EI, but both possess the remaining PTS proteins (Hengstenberg et al., 1969). Full PTS activity is restored when an exogenous source of HPr or EI is added to each mutant; in the presence of PEP, the EIIlac present in the extracts of S. aureus phosphorylates the chromogenic substrate o-nitrophenyl 
-D-galactoside (ONPG), yielding ONPG 6-phosphate. This compound is hydrolysed by the endogenous phospho--galactosidase, to give o-nitrophenol (ONP). The latter was determined by its absorbance at 420 nm.
For extract preparations, S. aureus, B. subtilis and B. sphaericus strains were grown to mid-exponential phase. Cells were harvested by centrifugation (10 000 g, 10 min), washed in 50 mM Tris/HCl pH 7·5, 10 mM DTT and 0·1 mM PMSF (buffer TTP) and recovered in the same buffer. After addition of lysozyme (10 mg ml-1 for B. subtilis and B. sphaericus) or lysostaphin (100 µg ml-1 for S. aureus) and incubation for 30 min at 37 °C, cells were broken. Approximately 0·2 g glass beads (100 µm and finer, Sigma) was added to each sample, and the sample was sonicated. After centrifugation (10 000 g, 15 min) to remove glass beads and cell debris, the supernatants were transferred to fresh tubes and used for both protein quantification and complementation reactions.
The reaction mixture contained, in 0·1 ml: 5 mM PEP, 10 mM MgCl2, 50 mM Tris/HCl, pH 7·5, 5 mM ONPG, 20 µl of S. aureus strain and 50 µl of the extract to be tested. Samples were incubated at 37 °C and reactions stopped by adding 0·9 ml 1 M Na2CO3. Each sample was centrifuged (12 000 g, 10 min) and the supernatant used to determine A420. Activity was expressed in Miller units (mg protein)-1.
Transport assays.
N-Acetylglucosamine (GlcNAc) transport studies were performed with cells grown in CTB minimal medium supplemented with either 0·5 % acetate or 0·5 % GlcNAc as described by Mobley et al. (1982). Cells were harvested during the exponential phase of growth (OD600 0·6–0·8), washed and resuspended in the same medium without the carbon source. Samples were kept on ice to maintain the PEP potential. A 1 ml sample of suspension was prewarmed at 32 °C for 5 min before addition of different concentrations of N-acetyl-D-[1-3H]glucosamine ([3H]GlcNAc; 8·30 Ci mmol-1, 37·4 mCi mg-1; Amersham [1 Ci=37 GBq]). At intervals from 30 s to 10 min, 0·1 ml aliquots were quickly mixed with 10 ml ice-cold phosphate buffer pH 7·0, filtered through 0·45 µm pore membrane filters (HAWP 02500, Millipore) and washed with 10 ml of the same ice-cold buffer. Filters were dried and the radioactivity measured with a scintillation counter.
In transport inhibition studies, the compound to be analysed was added at the concentration indicated 30 s before the addition of 50 µM [3H]GlcNAc. Samples were taken at 30 s intervals during 10 min and treated as described above.
Streptozotocin (Stz: [2d-2-(3-methyl-3-nitrosoureido)-D-glucopyranoside]) inhibition assays.
Cultures were pregrown for 16 h in LB (non-induced condition) or LB+0·5 % GlcNAc (induced condition), washed and diluted in fresh LB medium to OD600 0·12 and incubated for 60 min to initiate growth. They were then divided into three aliquots: one contained Stz 50 µg ml-1; a second contained Stz (50 µg ml-1) and 0·5 % GlcNAc; and a third had no supplement. The three cultures were incubated at 32 °C and samples withdrawn at different times to determine growth by measuring OD600.
Enzyme assays.
Studies of enzyme activities were performed with cells grown to OD600 0·6–0·8 in minimal medium (CTB) supplemented with either 0·5 % acetate or 0·5 % GlcNAc. Cultures were centrifuged, washed and resuspended in TTP buffer. Samples were sonicated in the presence of glass beads (Sigma) and centrifuged. Supernatants were assayed for glucosamine-6-phosphate deaminase (GlcN-6-phosphate deaminase; NagB) and N-acetylglucosamine-6-phosphate deacetylase (GlcNAc-6-phosphate deacetylase; NagA) activities as described previously (Bates & Pasternak, 1965). The assay mixtures in 0·5 ml contained: 50 mM Tris/HCl pH 7·4, 5 mM MgCl2, 5 U glucose-6-phosphate dehydrogenase (Sigma) and phosphoglucoisomerase (Sigma), 1 mM GlcNAc 6-phosphate (Sigma) [or GlcN 6-phosphate (Sigma) for GlcN-6-phosphate deaminase] and 0·3 mM NADP. The rate of NADP reduction was determined by measuring the rate of change in A340. Specific enzyme activities are given in nmol min-1 (mg protein)-1. Protein concentration was determined using a Bio-Rad kit with bovine serum albumin as standard.
Protein phosphorylation assays.
Cell extracts from B. sphaericus and B. subtilis were obtained as described above. The standard assay mixture for in vitro phosphorylation of HPr, contained in 20 µl, was: 50 mM Tris/HCl pH 7·5, 1 mM DTT, 5 mM NaF, 10 mM MgCl2, 2–5 µg HPr(His6) from B. subtilis or B. sphaericus or HPr(His6)S46Q, 2·5 mM (5 µCi) [
-32P]ATP (specific activity 109 c.p.m. µmol-1), 20 mM FBP and cell extract (5–50 µg protein). The mixtures were incubated at 32 °C or 37 °C for 5–15 min and reactions terminated by adding gel loading buffer and boiling for 3 min. Proteins were separated by 20 % or 17 % SDS-PAGE. Gels were boiled 5 min in the presence of 16 % TCA, dried and exposed to autoradiography film (Kodak Biomax MS) for 1–5 days at -70 °C.
[32P]PEP was synthesized from [-32P]ATP as described by Roossien et al. (1983). Phosphorylation of HPr(His6) from B. sphaericus was carried out in 30 µl of reaction mixture containing: 50 mM Tris/HCl pH 7·4; 10 mM MgCl2; 3 µl [32P]PEP; 0·5 µg EI(His6) of B. subtilis and 0·5 µg HPr(His6) of B. sphaericus. The reaction was incubated at 32 °C for 5–15 min and stopped by adding 5 µl gel loading buffer. The mixture was submitted to electrophoresis in 12·5 % SDS-PAGE. Gels were dried and exposed to autoradiography as described above.
RT-PCR assays.
Total RNA was isolated from cultures of B. sphaericus grown in LB or CTB supplemented with either 0·5 % acetate or 0·5 % GlcNAc using Trizol (Life Technologies) heated at 70 °C. The RNA was treated with RNase-free DNase (Promega) to eliminate contaminating DNA. For RT reactions, 10 µg total RNA was used for the synthesis of the first-strand cDNA using Moloney Murine Leukaemia (M-MLV) Virus Reverse Transcriptase enzyme (Ambion) with the indicated primer. The RT reactions (20 µl) were carried out according to the manufacturer's recommendations. Then 5 µl of each RT reaction mixture was used as template for PCR amplifications. Amplifications were carried out at the optimal annealing temperature for each pair of primers; products were visualized in 1–1·2 % agarose gels and analysed in a LAS 1000 Fuji analyser.
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; Mitchell et al., 1993; Titgemeyer et al., 1995) either B. subtilis or B. sphaericus extracts were able to complement both activities (Table 3). The values obtained with the different B. sphaericus homology groups were similar to those observed for B. subtilis 168 (heterologous positive control). These results clearly indicated that B. sphaericus had HPr- and EI-like proteins.
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, Fig. 1). In a PCR assay performed with DNA from B. sphaericus 2362, a fragment of the expected size was obtained. Its sequence was compared with databases and the 3' and 5' terminal regions showed similarity to ptsH and ptsI genes from other bacteria. The entire locus was then sequenced by inverse PCR (GenBank accession no. AY211495); the putative genes are shown in Fig. 1.
The ptsHI genes contained the sequences corresponding to His15 and His189 which were used for the first cloning. In addition, the last codon of ptsH overlapped the initiation codon of ptsI by 1 bp, suggesting an operon organization. Its structure is slightly different from that found in other Bacillus spp., such as B. subtilis (Gonzy-Tréboul et al., 1989), B. megaterium (Wagner et al., 2000) and B. stearothermophilus (Lai & Ingram, 1995), where the third base of the stop codon overlaps with the initiation codon. The HPr protein (88 amino acids and an estimated Mr of 9439) conserved the phosphorylation residues, His15 and Ser46, but it showed a low general identity when compared with other HPr proteins from low-G+C Gram-positive bacteria. However, several important residues (Asn43, Lys45, Ile47 and Met51) implicated in interactions with HPrK/P (Zhu et al., 1998) were conserved. On the other hand, the EI (570 amino acids and an estimated Mr of 62 528) showed high identity and similarity to other EI from low-G+C Gram-positive bacteria and the phylogenetic tree obtained groups it with other EIs from Bacillus spp. (data not shown).
Upstream of ptsH, another ORF (666 bp) was found whose termination codon also overlapped the second codon of ptsH. This arrangement suggested that the ptsHI operon could also include this new gene (nagB). This ORF would encode a protein (estimated Mr 24 651) which showed similarity to GlcN-6-phosphate deaminases (NagB) from different Gram-positive bacteria such as B. subtilis (O35000), Listeria monocytogenes (CAC99035) and Streptococcus agalactiae (AAM99686). Upstream of nagB, two genes whose ORFs suggest involvement in sugar utilization were observed: orf2 and nagA. The Orf2 protein (estimated Mr 26 464) showed similarity with several sugar isomerases, while NagA (estimated Mr 42 334) showed similarity to GlcNAc-6-phosphate deacetylase from B. subtilis (O34450), Bacillus halodurans (BAB040140), L. monocytogenes (CAC99034) and Vibrio furnisii (P96166S).
Functionality of HPr encoded by the cloned ptsH: mutagenesis and in vitro ATP- and PEP-dependent phosphorylation of HPr
To determine the functionality of the PTS general elements present in B. sphaericus, a ptsH mutant was obtained by insertional inactivation with a kan cassette (B. sphaericus ALE-7). Analysis by the in vitro complementation assay demonstrated that it lacked HPr activity (Table 3). This indicated that ptsH encoded a functional HPr.
In order to perform more detailed studies of the general PTS elements from B. sphaericus, His-tagged HPr [HPr(His6)] from B. sphaericus was cloned and expressed in E. coli, purified to homogeneity and used in phosphorylation assays. The in vitro phosphorylation assay was set up using B.subtilis EI(His6) and [32P]PEP as phosphate donor, and phosphorylation of B. sphaericus HPr(His6) could be observed (Fig. 2a). Then, a second assay was designed to determine if HPr(His6) from B. sphaericus could be phosphorylated at the Ser46 residue. Extracts of B. sphaericus and B. subtilis (positive control) were used as source of HPr kinase, with [32P]ATP as phosphate donor. B. sphaericus HPr(His6) and B. subtilis HPr(His6) were phosphorylated by B. subtilis extracts only in the presence of FBP (Fig. 2b: lanes 5 and 6, and lanes 7 and 8). FBP-dependent phosphorylation of B. sphaericus HPr(His6) could also take place using B. sphaericus cell extracts, demonstrating the presence of an HPr kinase activity in B. sphaericus. However, under our experimental conditions B. subtilis HPr(His6) could not be phosphorylated by B. sphaericus extracts. No modulatory effect of other compounds (i.e. gluconate 6-phosphate, fructose 6-phosphate) on HPrK activity could be observed (data not shown). The construction of a Ser46/Gln mutant and the failure of the corresponding HPr(His6)S46Q protein to be phosphorylated in similar assays (Fig. 2c), validated the importance of such Ser46 residue for kinase recognition.
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), including the ptsH mutant ALE-7 (not shown). Its thermal stability and size suggest that another small protein, perhaps Crh-like, could be phosphorylated by a kinase (possibly HPr kinase) and the supplied [-32P]ATP. However, further experiments would be required to ascertain its nature.
Characterization of GlcNAc metabolism and transport in B. sphaericus
The presence of nagB upstream of ptsHI, together with the fact that GlcNAc can be transported by the PTS in other micro-organisms (Plumbridge 1989; Peri & Waygood, 1988; Vogler & Lengeler, 1989; Reizer et al., 1999a), led us to consider GlcNAc as a likely candidate sugar to be transported and metabolized by B. sphaericus.
To investigate whether GlcNAc could be used as unique carbon source, B. sphaericus 2362 was grown in both the minimal medium described by Russell et al. (1989) and CTB medium with GlcNAc as sole carbon source. Although the doubling time was very long (120 min), strain 2362 was able to grow in those media without any significant difference. In contrast, the ptsH mutant strain ALE-7 failed to grow in CTB medium with GlcNAc as the sole source of carbon and energy. It also failed to complement PTS extracts from S. aureus mutants (Table 3). However, compounds such as glycerol, glutamate and gluconate normally utilized by wild-type strains (2362 and ASB13052) were also used by the ALE-7 mutant. No other differential phenotypic traits could be detected when the ptsH mutant was analysed using both APIB50 and APIZYM tests. This suggested that HPr was either involved in the transport and phosphorylation of GlcNAc (through a putative EIINag) or indirectly affected its transport and/or metabolism.
With the aim of elucidating the GlcNAc transport mechanism in B. sphaericus, [3H]GlcNAc uptake was studied. The assays were performed with cultures of strain 2362 grown in CTB with 0·5 % GlcNAc or acetate as carbon source. With cultures grown in acetate, a very low [3H]GlcNAc incorporation rate was observed compared with the GlcNAc cultures, suggesting that the transport system was inducible (data not shown). With GlcNAc-grown cultures, the kinetic parameters of the transport showed an affinity constant (Km) of 19 µM and maximum velocity rate (Vmax) of 1223 pmol min-1 mg-1, similar to other GlcNAc transporters of Gram-positive bacteria (Mobley et al., 1982; Wang et al., 2002).
In order to further analyse the uptake characteristics, several potential inhibitors were investigated. Compounds like carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin had no effect on GlcNAc transport (Table 4), indicating that this amino sugar was not transported by permeases coupled to either 
or pH. Iodoacetic acid was, however, a potent inhibitor (Table 4). Streptozotocin, an antibiotic which was reported to be transported through a GlcNAc PTS (PTSNag) (Ammer et al., 1979; Lengeler, 1980; Jacobson et al., 1990), was assayed. An inhibitory effect of Stz was observed in LB cultures pregrown in LB+GlcNAc. However, if the amino sugar was continuously present, Stz failed to inhibit growth (Fig. 3a), indicating that Stz and GlcNAc might compete for the same transporter but with different affinity. In cultures grown in LB without GlcNAc, Stz was slightly inhibitory (data not shown), indicating that induction was essential for antibiotic uptake and growth inhibition. Furthermore, in similar experiments with the ptsH mutant, Stz did not show any effect regardless of the presence or absence of GlcNAc in the LB medium (Fig. 3b). Altogether, these experiments suggest that a common transport system is used by GlcNAc and Stz. Because this transport required GlcNAc induction and a functional HPr, we think that it must be either a PTS permease or an EIINag. This last possibility was supported by the absence of inhibition with CCCP and monensin and the fact that other sugars and analogues tested, such as D-glucose, D-GlcN, 2-deoxy-D-glucose and methyl -glucoside, did not inhibit [3H]GlcNAc transport (Table 4). It is interesting to note that B. sphaericus 2362 cannot use D-GlcN as sole carbon source either.
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). Alternatively, the use of the Entner–Doudoroff pathway would require the presence of a glucose-6-phosphate dehydrogenase activity, which could not be detected in this or in previous works (Russell et al., 1989).
Transcription analysis of the nag–pts gene cluster by RT-PCR
To determine if the ptsH and ptsI genes were indeed co-transcribed, a RT-PCR was done with total RNA extracted from cells grown in LB. cDNA obtained with primer PtsI7, which is complementary to ptsI (Fig. 1), was used as template in a PCR with primers PtsHN and PtsHCH. A fragment with the expected size (275 bp) was amplified (Fig. 4a, lane 2), which demonstrated that the two genes are co-transcribed, as was observed in other micro-organisms.
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, lane 3). These observations strongly suggested that nagB, ptsH and ptsI are transcribed into the same mRNA.
Similar analyses were performed with nagA and orf2, but in this case, experiments were carried out with RNA extracted from cells grown in CTB with 0·5 % GlcNAc or 0·5 % acetate as carbon source. Fig. 4(b) shows the fragments amplified from cDNA obtained with the primer PtsI7 with different pairs of primers. In the presence of GlcNAc a long transcript spanning nagA, orf2, nagB, ptsH and ptsI was detected; however, in cells grown on acetate nagA mRNA was almost undetectable, indicating that nagA was poorly transcribed (Fig. 4b, lanes 5 and 6). These results were confirmed with cDNAs synthesized with primers O2 and NagA3 (Fig. 4c). Although the nagA mRNA was poorly detected in the long transcript in the presence of acetate, it could be unambiguously identified in the cDNA obtained with NagA3 (Fig. 4c, lanes 5 and 6). Although RT-PCR may not be a precise method for quantification of the transcription rates, an overview of these results indicates a clear tendency to get more intense amplification bands for nagA (and nagB) when RNA was extracted from GlcNAc-grown cells. This possible difference in expression due to the carbon source was not as clear for orf2 and ptsHI (Fig. 4d).
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have shown that in M. pneumoniae, HPrK/P phosphorylated the cognate HPr much more efficiently than HPr from B. megaterium. This was attributed just to the change in residue 48 (Ile in M. pneumoniae and Met in B. megaterium) required for the interaction between HPrK/P and HPr (Zhu et al., 1998; Himmelreich et al., 1996; Wagner et al., 2000). In this region, two amino acid sequence differences have been noticed between B. sphaericus HPr (Leu48 and Gly52) and B. subtilis HPr (Met48 and Ser52) that might explain the lack of B. sphaericus HPrK/P activity on B. subtilis.
A remarkable feature of these genes in B. sphaericus concerned their genetic structure: ptsHI were close to genes encoding GlcNAc catabolic enzymes (NagA and NagB). In B. subtilis, ptsG, encoding the EIIGlc, is sited upstream from ptsHI genes and the three genes are transcribed together in the presence of glucose (Stülke et al., 1997). In B. stearothermophilus another gene (ptsT) was found downstream of ptsHI (Lai & Ingram, 1995); however, its role has not yet been determined in this bacterium. In this work, we showed that in B. sphaericus, ptsHI are co-transcribed with nagA, orf2 and nagB, located upstream from ptsH. Transcription could be stimulated when cells were grown with GlcNAc. This stimulation seemed to affect particularly nagA (see Fig. 4a, b), suggesting the possibility of a second promoter for nagA or mRNA processing, or even multiple levels of control as reported for the glycolytic pathway in B. subtilis (Ludwig et al., 2001). Also the GlcNAc inducing effect was observed for NagA and NagB activities (Table 4). Induction of the nag operon by GlcNAc has also been reported in E. coli (Plumbridge, 2001).
The transport and metabolism of GlcNAc has mainly been studied in E. coli (Jones-Mortimer & Kornberg, 1980; Peri & Waygood, 1988; Plumbridge 1989, 1995) and B. subtilis (Clarke & Pasternak, 1962; Mobley et al., 1982; Reizer et al., 1999a) and the involvement of the PTS has been clearly established. In the present report, several lines of evidence indicate the contribution of the PTS. Also a ptsH mutant was shown to be unable to use GlcNAc as sole carbon source. The absence of any other phenotypic trait in this mutant suggested that in this micro-organism, the PTS was mainly implicated in GlcNAc transport and utilization. This would be supported by the arrangement of the genes. However, it cannot be excluded that other uncharacterized sugars may also be transported by this system.
In E. coli and other micro-organisms, GlcNAc and Stz compete for the PTSNag transporter (Ammer et al., 1979; Lengeler, 1980; Jacobson et al., 1990). Once transported into the cell, Stz is phosphorylated and becomes toxic. In B. sphaericus, Stz inhibited growth when cultures were pre-induced with GlcNAc, but Stz failed to inhibit when GlcNAc was simultaneously present, suggesting a competition for an EIINag transporter. Clearly, the ptsH mutant did not use GlcNAc and was insensitive to the antibiotic, indicating that neither compound was transported.
In E. coli, GlcNAc transported by EIINag is phosphorylated to GlcNAc-6P, which is metabolized to fructose 6-phosphate (F6P) through both GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase enzymes. F6P is then driven into glycolysis by the PFK enzyme. However, in Pseudomonas aeruginosa, a non-orthologue of nagB was found (Reizer et al., 1999b) and it was hypothesized that GlcN 6-phosphate was metabolized through the Entner–Doudoroff pathway as occurs in Pseudomonas fluorescens using glucosaminate as intermediate (Iwamoto & Imanaga, 1991). In B. sphaericus we have detected both nagB and nagA genes and their encoded activities; therefore, F6P may be formed during the metabolism of GlcNAc. Since neither glucose-6-phosphate dehydrogenase nor Entner–Doudoroff enzyme activities were detected in this bacterium (Russell et al., 1989; A. F. Alice, unpublished results), this pathway may not be functional in this species.
The existence of the PFK enzyme (Alice et al., 2002) together with the results shown here would constitute enough evidence to claim that GlcNAc could be metabolized through glycolysis. However, the existence of other route(s) cannot be discounted until the function(s) of the orf2-encoded protein is established.
Another interesting point is the fact that FBP, which is needed for the HPr kinase activity, would be synthesized during GlcNAc metabolism. The presence of an HPr kinase activity and a CcpA protein (Alice, 2001) would suggest that GlcNAc could trigger catabolite repression in B. sphaericus.
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Alice, A. F. (2001). Sistema de fosfotransferasa dependiente del fosfoenolpiruvato (PTS) y represión catabólica en Bacillus subtilis. PhD Thesis, University of Buenos Aires.
Alice, A. F., Perez-Martinez, G. & Sanchez-Rivas, C. (2002). Existence of a true phosphofructokinase in Bacillus sphaericus: cloning and sequencing of the pfk gene. Appl Environ Microbiol 68, 6410–6415.
Altschul, S. F., Madden, T. L., Schaeffer, A. A., Zhang, A., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3398–3402.
Ammer, J., Brennenstuhl, M., Schindler, P., Höltje, J. V. & Zähner, H. (1979). Phosphorylation of streptozotocin during uptake via the phosphoenolpyruvate : sugar phosphotransferase system in Escherichia coli. Antimicrob Agents Chemother 16, 801–807.
Bates, C. J. & Pasternak, C. A. (1965). Further studies on the regulation of amino sugar metabolism in Bacillus subtilis. Biochem J 96, 147–154.[Medline]
Baumann, P., Clark, M. A., Baumann, L. & Broadwell, A. H. (1991). Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins. Microbiol Rev 55, 425–436.
Charles, J. F., Nielsen-Le Roux, C. & Delécluse, A. (1996). Bacillus sphaericus toxins: molecular biology and mode of action. Annu Rev Entomol 41, 451–472.[CrossRef][Medline]
Charrier, V., Buckley, E., Parsonage, D., Galinier, A., Darbon, E., Jaquinod, M., Forest, E., Deutscher, J. & Clairbone, A. (1997). Cloning and sequencing of two enterococcal glpK genes and regulation of the encoded glycerol kinases by phosphoenolpyruvate-dependent, phosphotransferase system-catalyzed phosphorylation of a single histidyl residue. J Biol Chem 272, 14166–14174.
Clarke, J. S. & Pasternak, C. A. (1962). The regulation of amino sugar metabolism in Bacillus subtilis. Biochem J 84, 185–191.[Medline]
Couch, T. L. (2000). Industrial fermentation and formulation of entomopathogenic bacteria. In Entomopathogenic Bacteria: from Laboratory to Field Application, pp. 297–316. Edited by J. F. Charles and others. Dordrecht:: Kluwer.
deBarjac, H., Veron, M. & Cosmao Dumanoir, V. (1980). Caractérisation biochimique et sérologique de souches de Bacillus sphaericus pathogènes ou non pour les moustiques. Ann Microbiol 131B, 191–201.
Deutscher, J., Küster, E., Bergstedt, U., Charrier, V. & Hillen, W. (1995). Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to catabolite repression in Gram-positive bacteria. Mol Microbiol 15, 1049–1053.[Medline]
Dossonnet, V., Monedero, V., Zagorec, M., Galinier, A., Perez-Martinez, G. & Deutscher, J. (2000). Phosphorylation of HPr by the bifunctional HPr kinase/P-Ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion. J Bacteriol 182, 2582–2590.
Fujita, Y., Miwa, Y. Galinier A. & Deutscher, J. (1995). Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr. Mol Microbiol 17, 953–960.[CrossRef][Medline]
Galinier, A., Kravanja, M., Engelmann, R., Hengstenberg, W., Kilhoffer, M. C., Deutscher, J. & Hiech, J. (1998). New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression. Proc Natl Acad Sci U S A 95, 1823–1828.
Gonzy-Tréboul, G., Zagorec, M., Rain-Guian, M. C. & Steinmetz, M. (1989). Phosphoenolpyruvate : sugar phosphotransferase system of Bacillus subtilis: nucleotide sequence of ptsX, ptsH and the 5' end of the ptsI and evidence for a ptsHI operon. Mol Microbiol 3, 103–112.[CrossRef][Medline]
Guérout-Fleury, A. M., Shazand, K., Frandsen, N. & Stragier, P. (1995). Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167, 335–336.[CrossRef][Medline]
Haldenwang, W. G. (1995). The sigma factors of Bacillus subtilis. Microbiol Rev 59, 1–30.
Hengstenberg, W., Penberthy, W., Hill, K. & Morse, M. (1969). Phosphotransferase system of Staphylococcus aureus: its requirement for the accumulation and metabolism of galactosides. J Bacteriol 99, 383–388.
Henkin, T. M., Grundy, F. J., Nicholson, W. L. & Chamblis, G. H. (1991). Catabolite repression of -amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli LacI and GalR repressors. Mol Microbiol 5, 575–584.[CrossRef][Medline]
Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B. C. & Herrmann, R. (1996). Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res 24, 4420–4449.
Hoischen, C., Dijkstra, A., Rottem, S., Reizer, J. & Saier, M. H., Jr (1993). Presence of protein constituents of the Gram-positive bacterial phosphotransferase regulatory system in Acholeplasma laidlawii. J Bacteriol 175, 6599–6604.
Iwamoto, R. & Imanaga, Y. (1991). Direct evidence of the Entner–Doudoroff pathway operating in the metabolism of D-glucosamine in bacteria. J Biochem 109, 66–69.
Jacobson, G. R., Poy, F. & Lengeler, J. W. (1990). Inhibition of Streptococcus mutans by the antibiotic streptozotocin: mechanisms of uptake and the selection of carbohydrate-negative mutants. Infect Immun 58, 543–549.
Jault, J. M., Fieulaine, S., Nessler, S., Gonzalo, P., Di Pietro, A., Deutscher, J. & Galinier, A. (2000). The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding. J Biol Chem 275, 1773–1780.
Jones-Mortimer, M. C. & Kornberg, H. L. (1980). Amino-sugar transport systems in Escherichia coli K12. J Gen Microbiol 117, 369–376.[Medline]
Kravanja, M., Engelmann, R., Dossonnet, V. & 7 other authors (1999). The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase. Mol Microbiol 31, 59–66.[CrossRef][Medline]
Krych, V. K., Johnson, J. L. & Yousten, A. A. (1980). Deoxyribonucleic acid homologies among strains of Bacillus sphaericus. Int J Syst Bacteriol 30, 476–482.
Lai, X. & Ingram, L. O. (1995). Discovery of a ptsHI operon, which includes a third gene (ptsT), in the thermophile Bacillus stearothermophilus. Microbiology 141, 1443–1449.[Abstract]
Landt, O., Grunert, H. P. & Hanh, U. (1990). A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96, 125–128.[CrossRef][Medline]
Lengeler, J. (1980). Analysis of the physiological effects of the antibiotic streptozotocin on Escherichia coli K12 and other sensitive bacteria. Arch Microbiol 128, 196–203.[CrossRef][Medline]
Ludwig, H., Homuth, G., Schmalisch, M., Dyka, F. M., Hecker, M. & Stülke, J. (2001). Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon. Mol Microbiol 41, 409–422.[CrossRef][Medline]
Martin-Verstraete, I., Débarbouillé, M., Klier, A. & Rapoport, G. (1990). Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon. J Mol Biol 214, 657–671.[CrossRef][Medline]
Mijakovic, I., Poncet, S., Galinier, A. & 9 other authors (2002). Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life? Proc Natl Acad Sci U S A 99, 13442–13447.
Mitchell, W., Reizer, J., Herring, C., Hoischen, C. & Saier, M. H., Jr (1993). Identification of a phosphoenolpyruvate : fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes. J Bacteriol 175, 2758–2761.
Mobley, H. T., Doyle, R. J., Streips, U. N. & Langemeier, S. O. (1982). Transport and incorporation of N-acetylglucosamine in Bacillus subtilis. J Bacteriol 150, 8–15.
Peri, K. G. & Waygood, E. B. (1988). Sequence of cloned Enzyme IINag of the phosphoenolpyruvate : N-acetylglucosamine phosphotransferase system of Escherichia coli. Biochemistry 27, 6054–6061.[CrossRef][Medline]
Plumbridge, J. (1989). Sequence of the nagBACD operon in Escherichia coli K12 and pattern of transcription within the nag regulon. Mol Microbiol 3, 506–515.
Plumbridge, J. (1995). Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites. EMBO J 15, 3958–3965.
Plumbridge, J. (2001). Regulation of PTS gene expression by the homologous transcriptional regulators, Mlc and NagC, in Escherichia coli (or how two similar repressors can behave differently). J Mol Microbiol Biotechnol 3, 371–380.[Medline]
Porter, A. G., Davidson, E. W. & Liu, J. W. (1993). Mosquitocidal toxins of Bacilli and their genetic manipulation for effective biological control of mosquitoes. Microbiol Rev 57, 838–861.
Postma, P. W., Lengeler, J. W. & Jacobson, G. R. (1993). Phosphoenolpyruvate : carbohydrate phosphotransferase systems of bacteria. Microbiol Rev 57, 543–594.
Priest, F. G. (2000). Biodiversity of the entomopathogenic, endospore-forming bacteria. In Entomopathogenic Bacteria: from Laboratory to Field Application, pp. 1–22. Edited by J. F. Charles and others. Dordrecht: Kluwer.
Reizer, J., Peterkofsky, A. & Romano, A. H. (1988). Evidence for the presence of heat-stable protein (HPr) and ATP-dependent HPr kinase in heterofermentative lactobacilli lacking phosphoenolpyruvate : glycose phosphotransferase activity. Proc Natl Acad Sci U S A 85, 2041–2045.
Reizer, J., Hoischen, C., Titgemeyer, F., Rivolta, C., Rabus, R., Stülke, J., Karamata, D., Saier, M. H., Jr & Hillen, W. (1998). A novel protein kinase that controls carbon catabolite repression in bacteria. Mol Microbiol 27, 1157–1169.[CrossRef][Medline]
Reizer, J., Bachem, S., Reizer, A., Arnaud, M., Saier, M. H., Jr & Stülke, J. (1999a). Novel phosphotransferase system genes revealed by genome analysis – the complete complement of PTS proteins encoded within the genome of Bacillus subtilis. Microbiology 145, 3419–3429.
Reizer, J., Reizer, A., Lagrou, M. J., Folger, K. R., Kendall Stover, C. & Saier, M. H., Jr (1999b). Novel phosphotransferase systems revealed by bacterial genome analysis: the complete repertoire of pts genes in Pseudomonas aeruginosa. J Mol Microbiol Biotechnol 1, 289–293.[CrossRef][Medline]
), LB with Stz and GlcNAc (
), and LB ( 
); growth was monitored as OD600.