Comparative Analysis of Iron Regulated Genes in Mycobacteria

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Comparative Analysis of Iron Regulated Genes in Mycobacteria View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 580 (2006) 2567–2576 Comparative analysis of iron regulated genes in mycobacteria Sailu Yellaboina, Sarita Ranjan, Vaibhav Vindal, Akash Ranjan* Computational and Functional Genomics Group, Sun Centre of Excellence in Medical Bioinformatics, Centre for DNA Fingerprinting and Diagnostics, EMBnet India Node, Hyderabad 500076, India Received 9 January 2006; revised 20 March 2006; accepted 28 March 2006 Available online 12 April 2006 Edited by Robert B. Russell IdeR is a global regulator of iron response and belongs to the Abstract Iron dependent regulator, IdeR, regulates the expres- sion of genes in response to intracellular iron levels in M. tubercu- diphtheria toxin repressor (DtxR) family of transcription regu- losis. Orthologs of IdeR are present in all the sequenced genomes lators [7]. Electrophoretic mobility shift assay (EMSA) and of mycobacteria. We have used a computational approach to iden- DNA footprinting analysis have lead to the identification of tify conserved IdeR regulated genes across the mycobacteria and IdeR binding sites in upstream sequences of genes that code the genes that are specific to each of the mycobacteria. Novel iron the proteins that are involved in biosynthesis of siderophores regulated genes that code for a predicted 4-hydroxy benzoyl coA (MbtA, MbtB, MbtI), aromatic amino acids (PheA, HisE, hydrolase (Rv1847) and a protease dependent antibiotic regula- HisG), lipopolysacaharide molecules (Rv3402c), lipids (AcpP), tory system (Rv1846c, Rv0185c) are conserved across the myco- Peptidoglycan (MurB) and others annotated to be involved in bacteria. Although Mycobacterium natural-resistance-associated iron storage (BfrA, BfrB) [8,9]. DNA microarray analysis of macrophage protein (Mramp) is present in all mycobacteria, it is, iron-dependent transcriptional profiles of wild-type and IdeR as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-reg- mutant of M. tuberculosis has lead to the identification of variety ulated exochelin biosynthetic operon, which is present only in non- of other genes that code for the proteins like putative transport- pathogenic Mycobacterium, M. smegmatis. ers (Rv0282, Rv0283, Rv0284), members of the glycine-rich PE/ Ó 2006 Federation of European Biochemical Societies. Published PPE family (Rv2123), membrane proteins involved in virulence by Elsevier B.V. All rights reserved. (MmpL4, MmpS4), transcriptional regulators, enzymes in- volved in lipid metabolism (Rv1344, Rv1345, Rv1346, Keywords: Regulon; Modules; Prediction; Iron; Pathogen; Rv1347) and amino acid metabolism (TrpE2, PheA) [10]. Bacteria; Mycobacterium Orthologues of IdeR are present in all the sequenced gen- omes of mycobacteria. In this paper, we attempt to identify common and unique iron regulated genes in genomes of M. leprae, M. avium subsp. paratuberculosis and M. smegamatis. We applied a computational genomics tool – Predictregulon 1. Introduction to identify the IdeR binding motifs and operon context of that motif [11]. Previously reported IdeR binding sites from M. Iron is a cofactor for many enzymes and essential for growth tuberculosis were used to generate a recognition profile based of bacteria [1,2]. However, iron can also act as a potential cat- on Shannon relative entropy, which was used to predict poten- alyst of oxidative stress in bacteria. High amount of iron levels tial IdeR sites in sequenced genomes of mycobacteria. Further in a bacterium is countered by inducing synthesis of proteins we have also predicted the other co-expressed genes that are involved in iron storage and oxidative stress defense to protect potentially part of IdeR regulated operons. A sample of pre- against iron-mediated oxidative damage [3,4]. dicted motifs in M. smegmatis was experimentally verified by Iron limitation leads to the growth restriction of many spe- EMSA using recombinant M. tuberculosis IdeR. cies of mycobacteria including Mycobacterium tuberculosis, which causes tuberculosis in humans [5]. Iron is an obligate cofactor for at least 40 different enzymes encoded in the M. 2. Materials and methods tuberculosis genome. In pathogenic bacteria, many virulence factors and iron 2.1. In silico identification of IdeR binding sites acquisition systems are regulated by iron dependent transcrip- Published and annotated genome sequences of M. tuberculosis, M. tion regulators [6]. There are two such regulators identified in leprae and M. avium subsp. paratuberculosis were downloaded from NCBI ftp site (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/). Unpublished M. tuberculosis, ferric uptake regulator (furA) and Iron depen- genome sequence of M. smegmatis was downloaded from TIGR site dent regulator (IdeR). (http://pathema.tigr.org/tigr-scripts/CMR/CmrHomePage.cgi). The known IdeR binding sites collected from the literature [8,9] were used to built IdeR binding site recognition profile and identify the IdeR *Corresponding author. Fax: +91 40 27155610. binding sites as well as target genes in all the genomes of mycobacteria, E-mail address: [email protected] (A. Ranjan). using a method described previously [11,12]. Abbreviations: M. tuberculosis, Mycobacterium tuberculosis; M. avium subsp. paratuberculosis, Mycobacterium subsp. paratuberculosis; M. 2.2. Cloning, expression and purification of M. tuberculosis IdeR smegmatis, Mycobacterium smegmatis; DtxR, Diphtheria toxin repres- pQE30 expression vector (Qiagen) with an N terminal 6· His tag sor; IdeR, Iron-dependent regulator; RPS-BLAST, reversed position was used to clone the ORF Rv2711 of M. tuberculosis that encodes specific-basic local alignment search tool; EMSA, electrophoretic IdeR. Briefly, Rv2711 was taken out from pRSET IdeR construct mobility shift assay [13] with specific restriction enzyme sites (BamH1 and HindIII) and 0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.03.090 2568 S. Yellaboina et al. / FEBS Letters 580 (2006) 2567–2576 the insert was cloned into the corresponding sites of pQE30 expression Table 1 vector. Escherichia coli M15 cells transformed with the 6· His tagged Known IdeR binding sites from M. tuberculosis chimeric construct were grown in 400 mL of LB medium supplemented Binding site Gene with 100 lg/ml of ampicillin and 25 lg/ml of kanamycin. IPTG (0.2 mM) was added to a mid log phase culture. The cells were kept CAAGGTAAGGCTAGCCTTA Rv1519 in an incubator shaker for another eight hours at 27 °C and 200 rpm TTATGTTAGCCTTCCCTTA Rv3403c to allow protein expression. Then, cells were harvested by centrifuga- TTAACTTAGGCTTACCTAA Rv3839 tion and resuspended in 10 ml of lysis buffer (50 mM NaH2PO4, TTAGGCAAGGCTAGCCTTG Rv1343c 300 mM NaCl and 10 mM imidazole, pH 8) with 1 mM PMSF and CAAGGCTAGGCTTGCCTAA Rv1344 disrupted using a sonicator. After a second round of centrifugation TATGGCATGCCTAACCTAA Rv1347c for 10 min at 10000 · g, the supernatant was applied to a Ni–NTA TTCGGTAAGGCAACCCTTA Rv1348 affinity column (Qiagen, USA). The supernatant was allowed to bind ATAGGTTAGGCTACCCTAG Rv2122c to Ni–NTA column. The recombinant protein was eluted with CTAGGGTACCCTAACCTAT Rv2123 200 mM imidazole and analyzed by SDS–PAGE after washing the col- AGAGGTAAGGCTAACCTCA Rv3402c umn with 5 bed-volumes of wash buffer containing 20 mM imidazole. TTAGTGGAGTCTAACCTAA Rv1876 GTAGGTTAGGCTACATTTA Rv2386c 2.3. Electrophoretic mobility shift assay CTAGGAAAGCCTTTCCTGA Rv3841 Double-stranded oligonucleotides containing the predicted binding TTAGCTTATGCAATGCTAA Rv0282 motif (19 bp long) were end labeled with T4 polynucleotide kinase TTAGGCTAGGCTTAGTTGC Rv0451c and [32Pc]-ATP and were incubated with the purified recombinant TTAGCACAGGCTGCCCTAA Rv2383c IdeR protein in a binding reaction mixture. The binding reaction mix- TTAGGGCAGCCTGTGCTAA Rv2384 ture (20-ll total volume) contains the DNA-binding buffer (20 mM Tris–HCl [pH 8.0], 2 mM DTT, 50 mM NaCl, 5 mM MgCl2, 50% glyc- erol, and 5 lg of bovine serum albumin per ml), 10 lg of poly (dI–dC) per ml (for non-specific binding) and 200 lM NiSO4. The reaction mix- in DNA binding (Fig. 1). This suggests that the target DNA ture was incubated at room temperature for 30 min and loaded onto motifs in various genomes can be recognized based on se- 7% polyacrylamide gel containing 1· Tris–borate–EDTA buffer. No quence recognition profile generated from experimentally de- dye was added for loading. The gel was electrophoresed at 200 V for fined IdeR target motifs from M. tuberculosis. 2 h. Subsequently, the gel was dried and exposed to Storage Phosphor Image Plates for 4 h. The image plates were subsequently scanned in Storage Phosphor Imaging workstation. 3.2. In silico prediction of IdeR binding sites and target operons A recognition profile of experimentally defined IdeR binding sites (Table 1) from M. tuberculosis was used to identify the po- 3. Results and discussion tential IdeR binding sites and downstream operons/genes in genomes of M. avium subsp. paratuberculosis (Tables 2 and 3.1. IdeR from various species of actinobacteria shows a similar 3), M. smegamatis (Tables 4 and 5), M. leprae (Tables 6 and DNA binding domain 7). Function for the proteins encoded by these genes were pre- In order to assess the rationale of using M. tuberculosis IdeR dicted by RPS-BLAST (reversed position specific-basic local binding sites to identify the IdeR binding sites in
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