Department of Industrial Microbiology, Conway Institute of Biomolecular and Biomedical Sciences, University College Dublin, Dublin 4, and Dublin Molecular Medicine Centre, Dublin, Ireland1
Author for correspondence: Wim G. Meijer. Tel: +353 1 716 1364. Fax: +353 1 716 1183. e-mail: wim.meijer{at}ucd.ie
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ABSTRACT |
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Keywords: aceA, fadB, lactate and acetate metabolism, glyoxylate shunt, mRNA processing
a The GenBank accession number for the sequence reported in this paper is AY044917.
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INTRODUCTION |
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Virulence of R. equi is dependent on an 81 kb plasmid, indicating that this plasmid encodes virulence factors allowing the pathogen to establish itself in the host. Although the plasmid has been completely sequenced (Takai et al., 2000 ), it is still unknown what role the encoded proteins play in virulence. Although it has been firmly established that the virulence plasmid is essential for infection of foals (Takai et al., 1993
; Hondalus & Mosser, 1994
), it is clear that chromosomally encoded factors, such as regulatory genes (Boland & Meijer, 2000
), and metabolic pathways (Linder & Bernheimer, 1997
; Navas et al., 2001
) are equally important in allowing the pathogen to thrive within the host.
R. equi primarily infects alveolar macrophages of young foals and immunocompromised humans. Following uptake by macrophages, virulent R. equi strains grow rapidly in the phagolysosome (Hondalus & Mosser, 1994 ). At present it is poorly understood what sources of carbon, nitrogen and other essential nutrients are used during intracellular growth of this and other intracellular pathogens. Obvious sources of carbon are membrane cholesterol (Navas et al., 2001
) and lipids of the phagolysosome within which the pathogen resides. Lipids are generally dissimilated via beta-oxidation, resulting in the formation of acetyl-CoA, which is further metabolized via the tricarboxylic acid (TCA) cycle.
To assimilate acetate and lipids via acetyl-CoA, the two oxidative steps of the TCA cycle have to be bypassed. In most bacteria this is accomplished by the glyoxylate shunt, in which the subsequent activities of isocitrate lyase and malate synthase convert isocitrate and acetyl-CoA to succinate and malate. The relative flux of carbon through the assimilatory glyoxylate shunt or the dissimilatory TCA cycle is controlled by the relative activities of isocitrate lyase and isocitrate dehydrogenase, which compete for their common substrate isocitrate (Nimmo et al., 1987 ). It was recently shown that isocitrate lyase mutants of Mycobacterium tuberculosis and Candida albicans are strongly reduced in their ability to persist in mice or inflammatory macrophages. This emphasizes the importance of the glyoxylate shunt in long-term persistence in the host (McKinney et al., 2000
; Lorenz & Fink, 2001
) and supports the notion that membrane lipids may serve as a source of carbon for these and other intracellular pathogens.
The use of acetate and the potential use of membrane lipids by R. equi as carbon sources in soil and within macrophages, respectively, indicates that the glyoxylate shunt is important for the proliferation of both free-living and intracellularly located R. equi. To date virtually nothing is known regarding the glyoxylate shunt of R. equi. This paper shows that isocitrate lyase is maximally expressed following growth on acetate and lactate. The gene encoding isocitrate lyase was cloned and the transcriptional organization of this gene and fadB, encoding 3-hydroxyacyl-CoA dehydrogenase, was determined.
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METHODS |
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Media and growth conditions.
E. coli, R. fascians and R. equi strains were grown on LuriaBertani (LB) medium at 37 °C (Sambrook & Russell, 2000 ). Minimal R. equi medium consisted of (g l-1): K2HPO4, 5·0; NaH2PO4.H2O, 1·5; MgSO4.7H2O, 0·2; (NH4)2SO4, 1·0. Trace elements solution (0·2 ml; Vishniac & Santer, 1957
) and thiamine (0·1 mM) were added per litre of medium. Minimal medium was supplemented with acetate (20 mM), succinate (10 mM), lactate (20 mM) or pyruvate (20 mM). When appropriate, the following supplements were added: ampicillin, 50 µg ml-1; X-Gal, 20 µg ml-1; IPTG, 0·1 mM. Agar was added for solid media (1·5%, w/v).
DNA manipulations.
Plasmid DNA was isolated with the alkaline lysis method (Birnboim & Doly, 1979 ) or by using the Wizard Plus SV miniprep (Promega). Chromosomal DNA was isolated as described by Nagy et al. (1995)
. DNA modifying enzymes were used according to the manufacturers recommendations (Roche). Dideoxy sequencing reactions were done with the CEQ DCTS Kit as described by the manufacturer (Beckman). The nucleotide sequence was determined using a Beckman CEQ 2000 automatic sequencer; nucleotide sequence data were compiled using the Staden package (Staden et al., 2000
).
Southern hybridization.
Restriction digests of chromosomal DNA of R. equi were transferred to a positively charged nylon membrane as recommended by the manufacturer (Roche) following agarose gel electrophoresis. A 269 bp fragment representing an internal fragment of the isocitrate lyase gene of R. fascians (accession no. Z29367) was amplified by PCR with Pwo polymerase (Roche) using the oligonucleotides Rficfor (5'-AGGCTTCTTCGGTGTCAAGA-3') and Rficrev (5'-TGAACTGGAACTTGAAGCCC-3'). The reaction mixture was incubated at 94 °C for 5 min and was subsequently subjected to 30 cycles of 94 °C for 30 s, 55 °C for 45 s and 72 °C for 1 min, followed by an incubation at 72 °C for 5 min. The fragment was labelled with digoxigenin 11-dUTP using the DIG High Prime Kit (Roche). Prehybridization, hybridization and detection of the labelled probe were done according to the manufacturers recommendations.
RNA isolation.
Cells were harvested by centrifugation at 20000 g for 5 min; pellets were resuspended in 100 µl TES (10 mM Tris/HCl, pH 8, 1 mM EDTA, 1%, w/v, SDS) and incubated at 70 °C. The cell suspension was subsequently subjected to five cycles of incubation at 70 °C, followed by freezing in liquid nitrogen. RNA was isolated using the Promega SV RNA isolation kit according to the manufacturers instructions.
Northern hybridization.
Following electrophoresis on a denaturing formaldehyde gel (Sambrook & Russell, 2000 ), RNA was transferred to a positively charged membrane according to the manufacturers instructions (Roche). An internal fragment of the isocitrate lyase gene of R. equi was labelled by PCR amplification using the oligonucleotides Icl5 (5'-CAACGTCTACGAGCTGCAGA-3') and Icl10 (5'-TCGAACATGCCGTAGTTGAG-3') in the presence of 0·2 mM dATP, dCTP and dGTP; 0·13 mM dTTP and 0·07 mM digoxigenin 11-dUTP, using Taq polymerase according to the manufacturers instructions (Promega). The reaction mixture was incubated at 94 °C for 5 min and was subsequently subjected to 30 cycles of 94 °C for 30 s, 50 °C for 1 min and 74 °C for 1 min, followed by an incubation at 74 °C for 7 min. Prehybridization, hybridization and chemiluminescent detection of the labelled probe using DIG Easy Hyb and CDP-Star (Roche) were done according to the manufacturers recommendations. The relative abundance of transcripts was determined using a Fluor-S Max Multiimager System (Bio-Rad) and the Quantity One software package (Bio-Rad).
RT-PCR.
RT-PCR was carried out with the Promega Access RT-PCR System as per manufacturers instructions using mRNA isolated from acetate-grown R. equi. RNA corresponding to a 411 nt fragment was amplified using the oligonucleotides Icl24 (5'-ACCTGGCGCACGGCTACGCCC-3') and Icf2 (5'-GCTCGTACACCAGCACGTC-3'). The reaction mixture was incubated at 48 °C for 45 min and 94 °C for 5 min. It was subsequently subjected to 40 cycles at 94 °C for 30 s, 55 °C for 45 s and 68 °C for 1 min, followed by an incubation at 68 °C for 7 min. RNase-treated samples and reaction mixtures in which the AMV reverse transcriptase was omitted were used as control reactions.
Enzyme assays.
Cells were harvested at late exponential growth phase (OD430=1). Isocitrate lyase (EC 4.1.3.1) activity was determined at 37 °C in cell extracts (Dijkhuizen et al., 1978 ) by measuring the formation of glyoxylate phenylhydrazone in the presence of phenylhydrazine and isocitrate at 324 nm as described by Dixon & Kornberg (1959)
. Isocitrate dehydrogenase (EC 1 . 1 . 1 . 42) was determined at 37 °C by measuring the reduction of NADP at 340 nm (Levering & Dijkhuizen, 1985
). Protein was determined according to Bradford (1976)
using bovine serum albumin as standard.
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RESULTS |
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Nucleotide sequence of the aceA gene
Following nucleotide sequencing, one ORF preceded by a plausible ribosome-binding site was identified, which could encode a protein with a molecular mass of 46846 Da. Since the protein sequence specified by this ORF was 93% identical to that of R. fascians (Vereecke et al., 1994 ), the ORF was identified as aceA, encoding isocitrate lyase. The 5' end of a second ORF which was preceded by a ribosome-binding site and initiated with a GTG codon was identified 90 bp downstream of the aceA gene. The protein specified by this ORF was highly similar to FadB2, a 3-hydroxyacyl-CoA dehydrogenase from M. tuberculosis (66% identity). This ORF was therefore tentatively identified as fadB. Analysis of the 90 bp aceA-fadB intergenic region revealed the presence of a 17 bp inverted repeat, which may form a stable hairpin structure with a free energy (
G) of -28·5 kcal mol-1. A stretch of thymidines downstream from the hairpin structure, characteristic for
-independent transcriptional terminators (Platt, 1986
), was not present, indicating that this structure does not function as such.
aceA and fadB are cotranscribed
The small intergenic region between aceA and fadB strongly suggested that these genes are transcribed into a single transcript. To determine the transcriptional organization of this locus, mRNA was isolated from acetate-grown R. equi, during which growth the aceA gene is highly expressed. Northern hybridization, using an internal aceA fragment as probe, revealed two aceA transcripts of 2·8 and 1·6 kb (Fig. 2). Image analysis showed that the smaller transcript was 2·5 times more abundant than the larger transcript. The aceA gene is 1289 bp, whereas fadB genes are typically 858 bp. This indicates that the 2·8 kb transcript is at least bicistronic, encompassing both aceA and fadB. This was further examined by subjecting mRNA isolated from acetate-grown cells to RT-PCR using oligonucleotides complementary to aceA and fadB. A 411 bp product was observed encompassing the 3' end of aceA and the 5' end of fadB (data not shown), showing that these genes are cotranscribed.
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DISCUSSION |
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Surprisingly, the levels of isocitrate lyase and the ratio of isocitrate lyase and isocitrate dehydrogenase during growth on lactate were the same as during growth on acetate. In most bacteria lactate is metabolized to pyruvate by lactate dehydrogenase. Conversion of pyruvate to oxaloacetate by pyruvate carboxylase, or conversion of pyruvate to phosphoenolpyruvate followed by the formation of oxaloacetate by phosphoenolpyruvate carboxylase serve as anapleurotic reactions. These and other anapleurotic enzymes employed during growth on lactate enable the bacterium to assimilate carbon from lactate; isocitrate lyase is therefore not required for growth on lactate by bacteria employing lactate dehydrogenase (Meijer & Dijkhuizen, 1988 ). An explanation for the unexpectedly high activity of isocitrate lyase during growth on lactate could be that R. equi employs a lactate monooxygenase instead of lactate dehydrogenase. This enzyme, which is present in the closely related Mycobacterium smegmatis, converts lactate into acetate, carbon dioxide and water (Giegel et al., 1990
). If R. equi indeed employs lactate monooxygenase to metabolize lactate, then the glyoxylate shunt is required to assimilate acetate produced by this enzyme. The role of lactate monooxygenase in lactate metabolism of R. equi is currently under investigation in this laboratory.
Nucleotide sequencing showed that aceA is clustered with a gene similar to fadB, encoding 3-hydroxyacyl-CoA dehydrogenase, an enzyme participating in beta-oxidation of fatty acids. Analysis of aceA transcription using Northern hybridization showed the presence of two aceA mRNA species of 2·8 and 1·6 kb. The former transcript is sufficiently large to accommodate both aceA and fadB, which was confirmed by RT-PCR using oligonucleotides complementary to aceA and fadB. The presence of a smaller aceA mRNA, 2·5-fold more abundant than the aceA-fadB transcript, indicates that the aceA-fadB transcript is processed. The intergenic region between aceA and fadB contains a 17 bp inverted repeat capable of forming a stable hairpin structure. These structures are generally involved in stabilizing the upstream transcript by protecting it from 3'-5' exonuclease attack (Mclaren et al., 1991 ). The apparent aceA-fadB processing and the greater abundance of the aceA transcript indicates that this structure fulfils this role in R. equi.
The aceA and fadB genes are arranged in an identical manner in M. tuberculosis and, since they are separated by only 81 bp, are likely to form an operon. Genomic sequencing of the Streptomyces coelicolor genome (accession no. AL596102) showed that in this bacterium aceA is also clustered with fadB. However, in contrast to the mycolic-acid-containing actinomycetes, S. coelicolor harbours a malate synthase gene (aceB2) 100 bp downstream of aceA, followed by fadB. The small spacing between aceA and aceB2 and the fact that the stop codon of aceB2 and the start codon of fadB overlap, indicate that these three genes are cotranscribed as an operon.
Interestingly, in these three actinomycetes the aceA gene is separated from fadB (R. equi and M. tuberculosis) or aceB2-fadB (S. coelicolor) by an intergenic region of 81100 bp. Analysis of the 81 bp intergenic region between aceA and fadB2 and the 100 bp intergenic region between aceA and aceB2 revealed the presence of stable 17 bp (G=-24·5 kcal mol-1) and 15 bp (
G=-30·1 kcal mol-1) hairpin structures in M. tuberculosis and S. coelicolor, respectively. This strongly suggests that the putative aceA-fadB2 and aceA-aceB2-fadB transcripts in these bacteria are processed in a similar manner as in R. equi, leading to a stable aceA transcript. Isocitrate lyase is the first enzyme of the glyoxylate shunt and competes with isocitrate dehydrogenase for isocitrate. High isocitrate lyase activities are therefore required to create a sufficient flux of carbon through this pathway. In actinomycetes this seems to be accomplished at least in part by a stable aceA transcript which would allow high expression levels of this gene. Our current studies aim to further characterize mRNA processing of the aceA-fadB transcript of R. equi.
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ACKNOWLEDGEMENTS |
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Received 13 August 2001;
revised 16 October 2001;
accepted 29 October 2001.