Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000-Rosario, Argentina1
Author for correspondence: Diego de Mendoza. Tel: +54 341 4350596. Fax: +54 341 4390465. e-mail: diegonet{at}citynet.net.ar
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ABSTRACT |
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Keywords: Bacillus subtilis, cysteine biosynthesis, sulphate, transport
Abbreviations: TMS, transmembrane segment
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INTRODUCTION |
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Among bacterial sulphate transporters, those from Escherichia coli and Salmonella typhimurium are the best studied. These micro-organisms possess an ATP-binding cassette (ABC)-type sulphate-thiosulphate transport system (Higgins, 1992 ), which is controlled in parallel with cysteine biosynthetic enzymes and which is a part of the cysteine regulon; gene expression of this region requires sulphur limitation and a positive regulator, CysB, the product of the cysB gene (Kredich, 1996
). Components of the sulphate-thiosulphate permease from E. coli and S. typhimurium are encoded by the contiguous genes cysP, cysT, cysW and cysA, and by the unlinked gene sbp (Kredich, 1996
). The products of cysT and cysW span the membrane and form a channel for the passage of sulphate and related ions, cysA encodes a hydrophilic membrane-associated ATP-binding protein, and sbp and cysP encode the sulphate and thiosulphate periplasmic binding proteins, respectively (Hryniewicz et al., 1990
; Sirko et al., 1990
). Molecular analysis of sulphate transport in Synechococcus sp., a cyanobacterium, established a four-component membrane sulphate transport system, essentially similar to that of E. coli (Laudenbach & Grossman, 1991
).
The Neurospora crassa cys-14+ gene, the first eukaryotic sulphate transporter gene to be cloned, encodes a protein of approximately 90 kDa with 12 putative hydrophobic membrane-spanning domains (Ketter et al., 1991 ). The CYS14 protein is localized within the plasma membrane fraction and its synthesis depends on sulphur deprivation. Additional genes that encode H+/sulphate cotransporters have been identified in Homo sapiens (Hastbacka et al., 1994
), the tropical legume Stylosanthes hamata (Smith et al., 1995a
), Saccharomyces cerevisiae (Smith et al., 1995b
), Mus musculus (Kobayashi et al., 1997
), Arabidopsis thaliana (Takahashi et al., 1997
) and Yersinia enterocolitica (Hoffmann et al., 1998
). All of them show significant homology to each other and, together with CYS14, represent a family of membrane transport proteins, the sulphate permease (SulP) family.
In contrast to the knowledge of the sulphate transport system in Gram-negative bacteria and several eukaryotes, no information is available about the genes or the transport proteins involved in sulphate uptake in Bacillus subtilis or other Gram-positive organisms. We have recently reported the isolation of the B. subtilis cysH gene, whose product is 3'-phosphoadenosine 5'-phosphosulphate (PAPS) sulphotransferase and whose expression is repressed by cysteine and sulphide and induced by sulphur limitation (Mansilla & de Mendoza, 1997 ). A partial DNA sequence downstream of cysH revealed a second ORF, called ORF2, that gave a deduced amino acid sequence similar to that of a putative phosphate permease. As a result of the B. subtilis genome sequencing project, the complete nucleotide sequence of ORF2 was determined (Kunst et al., 1997
). This ORF was named ylnA; its gene product has 354 amino acids and it has a predicted molecular mass of 42·3 kDa. Both the cysH and ylnA genes form part of a 6074 bp putative operon containing seven ORFs (Fig. 1
). It has been demonstrated that three of these ORFs encode enzymes involved in cysteine biosynthesis: cysH encodes the PAPS sulphotransferase (Mansilla & de Mendoza, 1997
), whilst ylnD and ylnF encode proteins involved in synthesis of sirohaem, a cofactor of sulphite reductase (Johansson & Hederstedt, 1999
). ylnB and ylnC appear to be homologous to ATP-sulphurylase and adenosine 5'-phosphosulphate (APS) kinase, respectively, whilst ylnE does not show significant homology with any known protein contained in the databases.
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METHODS |
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Plasmid constructions.
Plasmid preparations, restriction enzyme digestions and agarose gel electrophoresis were carried out according to methods described by Sambrook et al. (1989) . Competent E. coli cells were transformed with supercoiled plasmid DNA by using the calcium chloride procedure or by electroporation (Sambrook et al., 1989
).
Expression analysis.
Recombinant plasmids containing the appropriate DNA fragments under T7 promoter control were transformed into E. coli BL21(DE3). Induction of gene expression was done as described by Studier et al. (1990)
. Briefly, mid-exponential-phase cultures (1 ml) of BL21(
DE3) carrying plasmids pBluescript II SK(+) or pBS181 were induced by adding 1 mM IPTG. After 10 min the cultures received rifampicin (100 µg ml-1) to inhibit mRNA synthesis from the E. coli chromosome. After further incubation for 1 h, they were labelled with 1 µCi [35S]methionine [specific activity 1000 Ci mmol-1 (37 TBq mmol-1)]. After 10 min, the cells were harvested by centrifugation, washed twice with 10 mM Tris/HCl buffer (pH 7·4), and stored frozen until analysis. Cells were resuspended in 30 mM Tris/HCl buffer (pH 7·4) containing 10 mM MgCl2 and 1 mM PMSF, and disrupted by sonication (four or five 10 s bursts) using a VibraCell Ultrasonic Processor (Sonics & Materials). The extracts were centrifuged at 190000 g for 1 h, and labelled proteins present in either soluble or membrane fractions were separated by SDS-PAGE and detected by autoradiography.
Assay of sulphate transport.
Cultures of strain JM2314 carrying relevant plasmids were grown in M9 minimal medium supplemented with glutathione as sulphur source to exponential phase. Cells were collected, washed and then resuspended in Davis salts (Miller, 1972 ) and incubated for 5 min at 30 °C. The measurement of sulphate transport was performed by incubating at 30 °C a cell suspension containing 108 cells ml-1, 30 µg chloramphenicol ml-1, 0·01 mM sodium sulphate and approximately 106 c.p.m.
ml-1 (1050 Ci mmol-1). The incubation period was terminated by filtering the cell suspension through a 0·45 µm Millipore filter. The filters were washed with 5 ml Davis salts. Filters were transferred to polyethylene vials containing 2 ml Optiphase HiSafe 3 scintillation fluid (Wallac) and the radioactivity counted in an LKB Primo liquid scintillation counter. Uptake rates are expressed in nmol sulphate min-1 (g cellular protein)-1.
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RESULTS AND DISCUSSION |
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Complementation analysis was performed with plasmid pBS170 (which contains cysH, ylnA and the 5' portion of ylnB, Table 1) and its derivatives: pBS181 (which includes a complete copy of ylnA), pBS184 (containing ylnA lacking the first 60 bp of the ORF), pBS188 (which includes ylnA without the last 140 bp of the ORF) and pBS190, a derivative of pBS181 possessing a frameshift mutation in ylnA (Table 1
). As shown in Fig. 2
, only plasmids pBS170 and pBS181, containing a complete functional copy of ylnA, restored the capacity of the cysA97, cysT and cysP sbp E. coli mutants to grow in minimal medium with sulphate as a sulphur source. These data strongly suggested that ylnA encodes a protein involved in sulphate transport. Moreover, the protein encoded by this gene should have a substrate recognition site since ylnA is able to bypass the requirement of E. coli for the sbp and cysP genes, which encode the sulphate and thiosulphate periplasmic binding proteins (Sirko et al., 1995
).
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We have found that sulphate is able to relieve the toxicity of chromate in cysP+ B. subtilis. In fact, strain JH642 is resistant to chromate in LB medium or in minimal media supplemented with 1 mM sulphate (data not shown). However, in minimal media supplemented with 1 mM glutathione as a sole sulphur source, the growth of strain JH642 is inhibited by chromate (data not shown). The toxicity of chromate in this case is relieved by the addition of 1 mM sulphate, thus indicating that sulphate by itself leads to reduced accumulation of chromate (data not shown). These results agree with the suggestion that the B. subtilis ChrA homologues YwrA and YwrB could catalyse a chromate/sulphate antiport, exchanging sulphate for the toxic accumulated chromate (Nies et al., 1998 ).
Expression of cysP in E. coli
To analyse the protein encoded by cysP, we used plasmid pBS181 containing cysP under the control of the T7 promoter (Table 1). This plasmid was transformed into E. coli BL21(
DE3). Induction of gene expression was done according to the method of Studier et al. (1990)
. Expression of the DNA insert containing cysP in pBS181 resulted in the detection of a protein of 24 kDa (Fig. 4
, lane 2). As described in the Introduction, the molecular mass calculated from the deduced primary sequence of CysP (YlnA) is 42·3 kDa, larger than the molecular mass of 24 kDa estimated by the mobility of the cysP gene product. Such an aberrant migration on SDS-PAGE is well documented for a variety of hydrophobic proteins (Buchel et al., 1980
; David et al., 1990
; Maiden et al., 1987
).
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Predicted structure of CysP
The hydropathy profile of the product of cysP, determined as described by Kyte & Doolittle (1982) , shows a hydrophobic peptide with 1012 transmembrane segments (TMSs) (Fig. 5
), resembling those of other membrane-associated proteins. Moreover, a putative cleavable N-terminal signal peptide, characteristic of membrane-bound proteins, was determined in CysP using the program PSORT (Nakai, 1996
).
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Analysis of the amino acid sequence of the cysP gene product using the BLAST algorithm (Altschul et al., 1997 ) revealed that this protein is composed of two homologous domains, called D1 and D2, possessing 69% similarity. Domain D1 extends from residue 1 to 150 and contains five TMSs (TMS 1TMS 5). Domain D2 extends from residue 163 to 313 and possesses TMS 6TMS 10. This topological organization of the transporter suggests that CysP might have arisen by a tandem internal gene duplication event.
Phylogenetic relationship
A comparison of the primary structure of CysP with protein sequences in the databases revealed a high level of similarity to those of several phosphate permeases of both prokaryotic and eukaryotic origin. However, no significant homology was found between CysP and proteins belonging to the SulP family.
Among the putative phosphate permeases, CysP shows a similar size and the same domain organization as the archaeal transporters (Table 2). The CysP gene product of B. subtilis showed a high level of similarity (6772%) to the putative phosphate transporters of Pyrococcus horikoshii, Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. We also found that Neurospora crassa Pho-4 and E. coli PitA proteins show significant homology with D1 in the N-terminal region and with D2 in the C-terminal region (Table 2
). Both proteins have a central region which is not homologous to CysP, which accounts for the difference in size, since both Pho-4 and PitA are larger than CysP (Mann et al., 1989
; Sofia et al., 1994
).
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Concluding remarks
The results presented here show that the cysP gene product is a membrane-bound protein with a gel electrophoresis migration corresponding to an apparent molecular mass of 24 kDa. The fact that cysP is able to restore the capacity to transport sulphate to the cysA97 strain JM2314 and to relieve cysteine auxotrophy of mutants affected in both the periplasmic and membrane components of the sulphate-thiosulphate permease of E. coli demonstrates that its gene product is a sulphate permease. It is interesting to note that in E. coli and S. typhimurium, sulphate and thiosulphate share the same periplasmic transport system (Kredich, 1996 ). However, in B. subtilis thiosulphate might be transported by a different permease, since strain MC2620, which possesses a Tn917 insertion downstream of cysH, exerting a polar effect on expression of cysP, is able to grow with thiosulphate as sole sulphur source (Mansilla & de Mendoza, 1997
).
The B. subtilis cysP gene encodes a novel membrane-associated sulphate transporter, without significant homology to sulphate transporters from both prokaryotic and eukaryotic organisms. This sulphate transporter belongs to a different class of carriers, the Pit family [Transport Commission (TC) no. 2.20; Paulsen et al., 1998 ]. Taking into account the homology of CysP to inorganic phosphate permeases, it remains to be determined if in addition to functioning as a sulphate permease, CysP is able to transport inorganic phosphate.
It is believed that proteins of the Pit family, as well as the members of other ancient families of secondary active transporters, such as the CaCA family (TC no. 2.19) and the Amt family (TC no. 2.49), are very restricted with respect to their substrate specificities (Paulsen et al., 1998 ). In this work we demonstrated that CysP is able to transport sulphate; thus, the assumed function of the uncharacterized members of the Pit family as putative phosphate transporters should be reconsidered.
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ACKNOWLEDGEMENTS |
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Received 2 September 1999;
revised 14 December 1999;
accepted 4 January 2000.