Departamento de Biología Molecular, Facultad de Medicina, Universidad de Cantabria, Unidad Asociada al Centro de Investigaciones Biológicas, CSIC, Cardenal Herrera Oria s/n, 39011 Santander, Spain1
Author for correspondence: Juan M. García-Lobo. Tel: +34 42 201948. Fax: +34 42 201945. e-mail: jmglobo{at}medi.unican.es
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ABSTRACT |
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Keywords: erythritol operon, Brucella abortus
Abbreviations: IHF, integration host factor
The GenBank accession number for the sequence reported in this paper is U57100.
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INTRODUCTION |
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The role of erythritol metabolism as a virulence factor remains obscure. This prompted us to undertake a genetic characterization of the metabolism of erythritol and its contribution to virulence in B. abortus. As a first step we generated a library of transposon insertion mutants of B. abortus strain 2308 as previously described (Sangari & Agüero, 1991 ). A mutant was isolated (mutant 227) that did not metabolize erythritol and was inhibited by this compound, mimicking the behaviour of the vaccine strain B19. This insertion mutant was used to clone the ery::Tn5 region from its chromosome, and then the corresponding regions from both strains 2308 and B19. By comparison of these regions a deletion was found in the chromosome of the B19 strain, allowing the development of a PCR assay for the rapid and unequivocal differentiation of the B19 vaccine strain from other Brucella strains (Sangari & Agüero, 1991
; Sangari et al., 1994
). The transposon insertion mutants in the ery genes were used to demonstrate that oxidation of erythritol was not essential for virulence in a mouse model of B. abortus infection (Sangari et al., 1998
). The aim of the work described here was to characterize the complete ery region from B. abortus 2308 and gain some insight into its regulation, thus enabling further analysis of the association between erythritol catabolism and virulence in Brucella.
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METHODS |
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Transposon mutagenesis.
Plasmid pSU6010 (Table 1), consisting of the complete ery region from B. abortus 2308 in a 7714 bp EcoRI fragment cloned in the broad-host-range, mobilizable vector pKT231 (Bagdasarian et al., 1981
), was able to complement the erythritol susceptibility of both B. abortus strain B19 and the ery::Tn5 mutant 227 (Sangari et al., 1994
). This plasmid was subjected to mutagenesis with the chloramphenicol-resistance transposon Tn1725 to map the position of ery genes precisely. To do this, pSU6010 was introduced into E. coli strain RU4404 (Ubben & Schmitt, 1986
), carrying Tn1725 in the chromosome. After 2 d growth at 30 °C, plasmid DNA was isolated and used to transform E. coli DH5
to chloramphenicol resistance. Plasmid DNA was purified from individual transformants and the location of the transposon was mapped with EcoRI and HindIII.
Construction of a B. abortus eryD mutant.
To construct a B. abortus eryD mutant by allelic replacement, we cloned a 2 kb NruIHindIII fragment containing eryD from the chromosome of strain 2308 (Sangari et al., 1994 ) into pBluescript SK. This plasmid was called pSU6101 (Fig. 1
). The central 0·5 kb BalI fragment from the eryD insert in pSU6101 was then replaced with a 1·2 kb kanamycin-resistance cassette from pUC4K. The resulting plasmid, pSU6103, contained a deleted eryD allele interrupted by the kanamycin-resistance cassette. The insert in pSU6103 was cloned into the plasmid pKOK.4, which can be transferred by conjugation into B. abortus, but which does not replicate in this species. In this way the plasmid pSU6106 was obtained and transferred into B. abortus 2308Nx. Kanamycin-resistant transconjugants were analysed by Southern blot hybridization with an eryD probe to check for replacement of the locus. One colony with the correct genomic structure (strain FJS-2) was selected for further work. The construction procedure is shown diagrammatically in Fig. 1
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Primer extension mapping of the 5' end of ery RNA.
Total RNA was prepared from 10 ml cultures as described previously (Garrido et al., 1993 ). For primer extension studies, RNA was used as template for the synthesis of cDNA by AMV reverse transcriptase (Boehringer Mannheim). Synthetic oligonucleotide primer M2990 (Table 2
) was 5' end-labelled with polynucleotide kinase and [
-32P]ATP. The products of the extension reactions were analysed in 6% urea-polyacrylamide gels. A sequencing ladder using the same primer was run in the gel beside the extension products to map the 5' end of the mRNA.
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RESULTS |
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The region between nucleotides 1 and 376 showed an A+T content much higher than the 41 mol% mean of the Brucella genome. A sequence identical to the integration host factor (IHF) binding site (Craig & Nash, 1984 ) was found in this region (116129). The sequence 252TTTACAN18TGTTAT281 (P1) was similar to the canonical E. coli promoter. Furthermore, some stretches of alternating As and Ts were suggestive of the existence of intrinsic curvature by analysis with the program DNASTAR (data not shown). All these data strongly suggested that this region could be involved in the transcriptional start and regulation of expression of downstream genes.
Four ORFs were identified in the region spanning basepairs 377 to 5400. The eryA gene could start either at the GTG codon at bp 377 or at the ATG at basepair 386. The presence of the sequence GAAAG, similar to the E. coli ribosome-binding site, 3 bp upstream of the GTG codon suggested that this was the initiation codon of eryA. The stop codon for this gene was found at position 1934. Translation of this sequence would result in a 519 aa polypeptide with a calculated molecular mass of 54·4 kDa. The next gene in the sequence (eryB) would start at the ATG at basepair 1949 and its stop codon was found at basepair 3455. This gene would translate into a 502 aa polypeptide with a calculated molecular mass of 56·2 kDa. The eryC gene started at 3465 and ended at 4392. Its product would be 309 aa long with a molecular mass of 35 kDa. Finally, eryD was found between positions 4422 and 5370; its translated product would be a protein of 316 aa with a molecular mass of 33·5 kDa. Genes eryA, eryB and eryC were closely spaced with very short intergenic regions in which sequences resembling the E. coli ribosome-binding site were found, and there were only 30 bp between eryC and eryD. A sequence found 3' to eryD between basepairs 5372 and 5412 was able to fold as a stemloop structure and had some analogies with transcription terminators. These data suggested that the four genes eryABCD could constitute a single transcriptional unit. Three additional genes, encoding a triose phosphate isomerase, a subunit of the galactose-6-phosphate isomerase and a regulatory protein belonging to the DeoR family, were found in the sequence downstream of the ery operon.
Analysis of the ery gene products by sequence similarity
The relationship of the polypeptides deduced from the nucleotide sequence of the ery region to known gene products was determined using the program TBLASTN. The results of these comparisons are summarized in Table 3.
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EryB was found to be a homologue of aerobic glycerol-3-phosphate dehydrogenases. The greatest identity (50·4%) was with the enzyme from E. coli. EryB had the PROSITE signature of FAD-dependent glycerol-3-phosphate dehydrogenases, PS00977. These findings and the structural similarity between glycerol phosphate and erythritol phosphate suggested that EryB was the erythritol phosphate dehydrogenase needed for the catabolism of erythritol.
We failed to find any protein in the database with extended similarity to the complete EryC. The most significant match corresponded to a limited region (57 aa) of the large subunit (HupL) of Alcaligenes hydrogenophilus hydrogenase (accession number S56898).
EryD showed similarity to several DNA-binding regulatory proteins. The closest homologue of EryD was SmoC, the regulator of a Rhodobacter sphaeroides operon involved in polyol transport and metabolism. Residues 2142 of EryD were identified with a 71% probability as a helixturnhelix motif using the method described by Dodd & Egan (1990) . This finding suggested that EryD was a DNA-binding protein with a regulatory function.
Identification of the ery operon promoter
Functionality of the putative promoter identified by sequence analysis was studied by primer extension. Synthetic oligonucleotide M2990 (Table 2), complementary to bases 314335 of the ery sequence, was used to identify the transcriptional start point. The results of the primer extension study are shown in Fig. 3
. A major band was obtained whose size indicated that the putative transcriptional start point was the residue G291, located 10 bp downstream of the promoter. This result confirmed that P1 was a functional promoter that directs the transcription of the ery operon.
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Expression of ery genes in E. coli
Since wild-type E. coli cells are unable to utilize erythritol we introduced recombinant plasmids containing different genes from the ery operon into E. coli HB101 and studied the ability of the recombinants to use erythritol. Using this assay and the set of plasmids described in Fig. 4, we determined that the minimal region required for degradation of erythritol in E. coli was eryABC (Fig. 4
). In addition, plasmid pSU6004 was introduced into several E. coli strains to investigate the effects of various E. coli genes on ery expression. Strain MC296, a himA hip (IHF-) double mutant did not utilize erythritol upon introduction of pSU6004, whereas the isogenic strain with the wild-type IHF alleles (MC294) did. This finding correlated with the presence of an IHF-binding site in the ery control region and strongly suggested a role for IHF in the regulation of expression of the ery operon. E. coli DH5
also produced white colonies on McConkey-erythritol plates when transformed with the ery plasmids. The relA mutation could be responsible for the poor expression of the ery operon in this strain.
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DISCUSSION |
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We have been unable to assign a function to EryC by sequence comparison. A clue to EryC function was obtained upon analysis of the ery operon in the vaccine strain B. abortus B19, which is the only known B. abortus isolate whose growth is inhibited by erythritol. The metabolism of erythritol in B. abortus B19 was studied biochemically by Sperry & Robertson (1975a) , who found that this strain lacks D-erythrulose-1-phosphate dehydrogenase activity. We have previously described the presence of a deletion in the B19 chromosomal ery locus (Sangari et al., 1994
). The results of the present study show that the deletion in B19 is located between positions 3975 and 4676. It affects two genes, eryC and eryD. As result of the deletion, 137 aa at the carboxyl end of EryC and 85 aa at the amino end of EryD, including its putative DNA-binding domain, are lost. A nonfunctional fused polypeptide containing the amino half of EryC and the carboxyl end of EryD could be produced in B19. Thus the B. abortus B19 strain is an eryCD double mutant. The defect in B19 was complemented in trans by plasmids containing the complete ery region and by plasmids with Tn1725 insertions in eryA, eryB and eryD. Plasmids with Tn1725 insertions in eryC were the only ones that failed to complement the Ery- phenotype of B19 (Fig. 2
). This indicated that the product of the eryC gene could be the enzyme D-erythrulose-1-phosphate dehydrogenase.
Sequence analysis and comparison showed that EryD was a protein with a helixturnhelix motif and probably possessed some regulatory function. To obtain further evidence for this, we constructed an eryD mutant (FJS-2) and compared the levels of ery transcription in this mutant and a wild-type strain. Transcription from the ery promoter (P1) was more active in both the mutant FJS-2 and strain B19 than in strain 2308. This result strongly suggested a repressor function for EryD. The increase in ery mRNA level induced by erythritol in strain 2308 indicated an inducing function for erythritol, probably through binding to EryD. Many transcription repressors with a helixturnhelix motif bind sugars that function as inducers. In addition to control by EryD, we have found an IHF-binding site upstream of the P1 promoter and results suggested that IHF was required for ery expression in E. coli. Although IHF has not yet been described in Brucella, it is generally accepted that all bacteria must contain a protein of this type. The role of IHF as a transcriptional regulator in other species has been well documented (Perez-Martin et al., 1994 ).
The genes in the ery operon described here encode three enzymes that are able to transform erythritol into 3-keto-L-erythrose 4-phosphate according to the pathway described by Sperry & Robertson (1975a) . E. coli cells provided with these three enzymes were capable of erythritol utilization. This observation suggests that the enzymes for degradation steps beyond 3-keto-L-erythrose 4-phosphate may be organized in other pathways, and their genes found at other chromosomal locations. Since this is the first description of an operon for erythritol utilization, comparisons are not possible. However, the analysis of operons for polyol utilization (Heuel et al., 1998
) highlights the lack of a gene for erythritol transport. Erythritol was shown to be a substrate for the E. coli glycerol facilitator GlpF (Heller et al., 1980
). Furthermore it is known that B. abortus B19 mutants tolerant to erythritol arise frequently, and that those mutants show impaired ability to grow on glycerol culture media (Sangari et al., 1996
). Thus it is possible that erythritol is transported into Brucella through a GlpF analogue, and later phosphorylated by erythritol kinase, trapping the polyol in the bacterial cytoplasm. The results reported here explain the utilization of erythritol by B. abortus, as well as the inhibition of growth in strain B19 by overproduction of the kinase EryA and the dehydrogenase EryB. The growth-promoting effect of erythritol has not been addressed in this work but there could be a link between erythritol and the aromatic pathway. Erythrose 4-phosphate, the precursor for the biosynthetic pathway for aromatic compounds in bacteria, may be easily obtained from the intermediates of erythritol catabolism. Thus erythritol may be crucial to obtain molecules such as aromatic amino acids and catechols, which play important physiological roles in Brucella, providing a connection between erythritol metabolism and virulence in B. abortus.
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ACKNOWLEDGEMENTS |
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Received 21 June 1999;
revised 10 October 1999;
accepted 1 November 1999.